Dioxolane analogues of cytidine for the treatment of cancer

ABSTRACT

The invention provides compounds of the formula: 
     
       
         
         
             
             
         
       
         
         R 1  is OR 11 , or NR 5 R 5′ ; 
         R 2  is H or F; 
         R 5  is H, OH, C 1 -C 6  alkyl, OH, C(═O)R 6 , O(C═O)R 6  or O(C═O)OR 6 ; 
         R 5′  is H or C 1 -C 6  alkyl; 
         R 6  is C 1 -C 6  alkyl or C 3 -C 7  cycloalkyl; 
         R 13  is H, phenyl, pyridyl, benzyl, indolyl or naphthyl wherein the phenyl, pyridyl, benzyl, indolyl and naphthyl is optionally substituted with 1, 2 or 3 R 22 ; 
         and the other variables are as defined in the claims, 
         which are of use in the treatment of cancer, and related aspects.

This application is the National Phase Under 35 USC § 371 of PCTInternational Application No. PCT/EP2015/069370 filed on Aug. 24, 2015,which claims priority under 35 U.S.C. § 119 on Patent Application No.1450983-0 filed in Sweden on Aug. 25, 2014, and Patent Application No.1550858-3 filed in Sweden on Jun. 22, 2015, the entire contents of eachof which are hereby incorporated by reference.

TECHNICAL FIELD

The present invention relates to phosphorus prodrugs of troxacitabineand derivatives thereof which are useful in the treatment of cancers, inparticular liver cancer such as hepatocellular carcinoma (HCC) andsecondary liver cancers. The invention further relates to compositionsand combinations comprising these compounds, and methods for their usein the treatment of cancers, particularly liver cancer such as HCC.

BACKGROUND TO THE INVENTION

Primary liver cancer is the sixth most frequent cancer globally and thesecond leading cause of cancer death. The most frequent liver cancer,accounting for approximately 85% of all primary malignant liver cancersand has a rising incidence, is hepatocellular carcinoma (HCC), which isformed by hepatocytes that become malignant. Another type of cancerformed by hepatocytes is hepatoblastoma, a rare malignant tumour thatprimarily develops in children, and accounts for approximately 1% of allcancers in children and 79% of all primary liver cancers under the ageof 15. Secondary liver cancer, or metastasis in the liver, is a cancerthat starts somewhere else in the body and then spreads to the liver.Examples of secondary liver cancer includes many common types of cancer,such as colon, rectum, lung, and breast cancer. Liver cancer can alsoform from other structures within the liver such as the bile duct, bloodvessels and immune cells. Cancer of the bile duct (cholangiocarcinomaand cholangiocellular cystadenocarcinoma) account for approximately 6%of primary liver cancers.

While surgical resection and liver transplantation are potentiallycurative therapies for early stage HCC, more than 20% of the patientswill eventually relapse or encounter further problems, and the majorityof HCC diagnosis take place at a stage that is too advanced for thesetreatments. Regional therapies, such as radiofrequency ablation areassociated with response rates above 60%, but they are only suitable fora certain proportion of patients and are not always curative.Chemotherapy used so far has been minimally effective in HCC and to dateresponse rates have not exceeded 25%. At present, sorafenib is the onlyeffective drug on the market for the treatment of advanced orunresectable HCC, therefore, there is a great need for furthertreatments of HCC to reduce relapse rates and increase overall survivalrates.

Many nucleoside analogues have been found to possess anticancer activityand they constitute a major class of chemotherapeutic agents that arewidely used for the treatment of patients with cancer. This group ofagents, known as antimetabolites, includes a variety of pyrimidine andpurine nucleoside derivatives with cytotoxic activity.

Cellular nucleotide kinases phosphorylate nucleosides to theircorresponding 5′-monophosphates which are further converted into theirdiphosphate and subsequently to the pharmacologically activetriphosphate. It is known that some nucleosides are weakly activebecause they cannot be efficiently phosphorylated by kinases or are notsubstrates for kinases at all. In the phosphorylation sequence, thefirst phosphorylation of nucleoside analogues is rate limiting whereasthe second and third phosphorylations are less sensitive tomodifications to the nucleoside. Nucleoside monophosphates (nucleotides)per se are generally unstable in blood and show poor membrane permeationand hence are not suitable for use as drugs. Due to the high instabilityand poor cellular permeation of triphosphate of nucleosides andnucleoside analogues they cannot either be considered as possible drugcandidates.

Troxacitabine, (beta-L-dioxolane cytidine) is a cytotoxic deoxycytidineanalogue with an unnatural L-configuration which has demonstrated broadactivity against both solid and hematopoietic malignancies in vitro andin vivo. Particularly, impressive activity has been observed againsthuman cancer cell lines and xenografts of hepatocellular, prostate, andrenal origin (Cancer Res., 55, 3008-3011, 1995). Troxacitabine has shownto give rise to a mutation of the kinase deoxycytidine kinase (dCK)which is normally responsible for the first phosphorylation step of thenucleoside, leading to no or very low levels of troxacitabinemonophosphate, thereby leading to resistance.

Troxacitabine entered phase III clinical trials in 2008 in the acutemyologenous leukemia indication, but did not proceed to registration.Discontinued phase II trials with troxacitabine include breast cancer,colorectal cancer, pancreatic cancer, melanoma, NSCLC, renal, prostateand ovarian tumours. Troxacitabine was generally administered as anintravenous infusion, thereby exposing many tissues to the drug,irrespective of the site of the cancer.

It has been shown that troxacitabine, despite its hydrophilic character,is transported into cells by passive diffusion, but is only very slowlyaccumulated in cancer cells in comparison with other, carriertransported nucleosides.

In WO2008/030373 derivatives of troxacitabine carrying a prodrug groupon the cytosine base moiety are disclosed and the relationship betweenthe lipophilicity of the prodrugs and their antitumor activity isevaluated. The patent states that base modification is desirable toavoid esterase difficulties with 5′-OH modification.

Phosphoramidate prodrugs at the 5′ hydroxyl function of D-nucleosideshave been successfully employed in antiviral drugs, such as sofosbuvirused in the treatment of HCV infection.

Unmasking of the sofosbuvir prodrug to reveal the monophosphateintracellularly is a complex, multistep process involving severalhydrolase enzymes in a particular sequence.

The use of phosphoramidate prodrugs on cancer nucleosides has been lesssuccessful. Nucana is developing Acelerin (Nuc-1031), a phosphoramidateprodrug of the D-nucleoside gemcitabine for the treatment of pancreaticcancer (for structure: see page 71 of WO2005012327). However, eventhough the phosphoramidate would be thought to enhance lipophilicity andcell permeability of the compound, the Acelarin prodrug must still beadministered as an IV infusion, thus exposing many healthy tissues tothe cytotoxic metabolite.

There is even less experience with monophosphate prodrugs ofL-nucleosides such as troxacitabine. WO2008048128 discloses a smallnumber of troxacitabine monosphosphate prodrugs including the compoundat Example 14:

No cancer or other biological activity is disclosed for any of thecompounds, either in the WO2008048128 specification or elsewhere in theacademic literature. There are no reports of such a prodrug enteringclinical trials. However, the inventors of WO2008048128 have publishedbroadly similar prodrugs on the D-nucleoside gemcitabine (Baraniak et alBiorg Med Chem 2014 2133-2040) where the prodrug approach appears towork in certain tissues, and the D-nucleoside azidothymidine (Kulic etal Antivir Chem Chemother 2011 21(3) 143-150) where the prodrugs were2-20 times less potent than the corresponding parent nucleoside. Kulicspeculates that the azidothymidine prodrugs tended to be firstdephosphorylated to the nucleoside and only then phosphorylated to theactive triphosphate species. In that the prodrug approach works ongemcitabine (which resembles RNA by virtue of its substituted 2′function), and does not work on azidothymidine (which is 2′-deoxythereby resembling DNA), it is hypothesised that the WO2008048128prodrugs of troxacitabine (which is a DNA analog, albeit L-DNA), arelikely to be inactive, like the azidothymidine prodrugs.

Balzarini et al Biochem Biophys Res Comm 225, 363-369 (1996) describethe HIV and HBV activity of CF 1109, a phosphoramidate prodrug of theL-nucleoside lamivudine/3TC, having the structure:

Balzarini states that this phosphoramidate prodrug was ˜250 fold lessactive against HIV than its parent nucleoside 3TC, but that the prodrugas “virtually equally effective against HBV in Hep G2.2.15 cells”. Inother words, addition of this large, phosphoramidate methyl esterprodrug, group did not improve antiviral potency in a liver cell line.Balzarini did not assay whether the prodrug was being metabolized to 3TCprior to being phosphorylated to the active triphosphate.

The present invention provides phosphorus prodrugs of troxacitabine,particularly liver targeted prodrugs such as phosphoramidates, which aresuitable for oral administration. These prodrugs have the advantage ofimproved cell permeability due to increased lipophilicity compared totroxacitabine per se, and to more efficient form the active triphosphatedue to bypassing the rate limiting first phosphorylation step. Further,the compounds of the invention are primarily metabolised to the activetriphosphate in the liver thereby providing a high concentration ofactive compound in the target organ and at the same time keeping sideeffects due to toxicity in other organs to a minimum.

DESCRIPTION OF THE INVENTION

In one aspect, the present invention provides compounds represented byFormula (I):

wherein:

R¹ is OR¹¹, or NR⁵R^(5′);

R² is H or F;

R⁵ is H, C₁-C₆alkyl, OH, C(═O)R⁶, O(C═O)R⁶ or O(C═O)OR⁶;

R^(5′) is H or C₁-C₆ alkyl;

R⁶ is C₁-C₂₂alkyl or C₃-C₇ cycloalkyl;

R¹¹ is H or C₁-C₇ alkyl;

R¹³ is H, phenyl, pyridyl, benzyl, indolyl or naphthyl wherein thephenyl, pyridyl, benzyl, indolyl and naphthyl is optionally substitutedwith 1, 2 or 3 R²²;

R¹⁵ is H, C₁-C₆ alkyl, C₃-C₇ cycloalkyl, C₃-C₇ cycloalkyl C₁-C₃ alkyl,phenyl, benzyl or indolyl;

R^(15′) is H or C₁-C₆ alkyl; or

R¹⁵ and R^(15′) together with the carbon atom to which they are attachedfrom a C₃-C₇ cycloalkylene group, wherein each C₁-C₆alkyl is optionallysubstituted with a group selected from halo, OR¹⁸ and SR¹⁸, and eachC₃-C₇ cycloalkyl, C₃-C₇ cycloalkylene, phenyl and benzyl is optionallysubstituted with one or two groups independently selected from C₁-C₃alkyl, halo and OR¹⁸; R¹⁶ is H, C₁-C₁₀ alkyl, C₂-C₁₀ alkenyl, C₃-C₇cycloalkyl, C₃-C₇ cycloalkyl C₁-C₃ alkyl, benzyl, or phenyl, any ofwhich is optionally substituted with 1, 2 or 3 groups, eachindependently selected from halo, OR¹⁸ and N(R¹⁸)₂;

each R¹⁸ is independently H, C₁-C₆ alkyl, C₁-C₆ haloalkyl or C₃-C₇cycloalkyl;

each R²² is independently selected from halo, C₁-C₆ alkyl, C₂-C₆alkenyl, C₁-C₆ haloalkyl, C₁-C₆ alkoxy, C₁-C₆ haloalkoxy, phenyl,hydroxyC₁-C₆ alkyl, C₃-C₆ cycloalkyl, C₁-C₆ alkylcarbonyl, C₃-C₆cycloalkylcarbonyl, carboxy C₁-C₆ alkyl, hydroxy, amino CN, and NO₂, orany two R²² groups attached to adjacent ring carbon atoms can combine toform —O—(CR²³R^(23′))₁₋₆—O—;

R²³ and R^(23′) are independently H or C₁-C₃ alkyl;

or a pharmaceutically acceptable salt and/or solvate thereof.

In one embodiment, the invention provides compounds represented byformula I:

wherein:

R¹ is OR¹¹, or NR⁵R^(5′);

R² is H or F;

R⁵ is H, C₁-C₆ alkyl, OH, C(═O)R⁶, OC(═O)R⁶ or OC(═O)OR⁶;

R^(5′) is H or C₁-C₆ alkyl;

R⁶ is C₁-C₂₂ alkyl or C₃-C₇ cycloalkyl;

R¹¹ is H or C₁-C₆ alkyl;

R¹³ is H, phenyl, pyridyl, benzyl, indolyl or naphthyl wherein thephenyl, pyridyl, benzyl, indolyl and naphthyl is optionally substitutedwith 1, 2 or 3 R²²;

R¹⁵ is H, C₁-C₆ alkyl, C₃-C₇ cycloalkyl, C₃-C₇ cycloalkyl C₁-C₃ alkyl,phenyl, benzyl or indolyl;

R^(15′) is H or C₁-C₆ alkyl; or

R¹⁵ and R^(15′) together with the carbon atom to which they are attachedfrom a C₃-C₇ cycloalkylene group, wherein each C₁-C₆alkyl is optionallysubstituted with a group selected from halo, OR¹⁸ and SR¹⁸, and eachC₃-C₇ cycloalkyl, C₃-C₇ cycloalkylene, phenyl and benzyl is optionallysubstituted with one or two groups independently selected from C₁-C₃alkyl, halo and OR¹⁸;

R¹⁶ is H, C₁-C₁₀ alkyl, C₂-C₁₀ alkenyl, C₃-C₇ cycloalkyl, C₃-C₇cycloalkyl C₁-C₃ alkyl, benzyl, or phenyl, any of which is optionallysubstituted with 1, 2 or 3 groups, each independently selected fromhalo, OR¹⁸ and N(R¹⁸)₂;

each R¹⁸ is independently H, C₁-C₆ alkyl, C₁-C₆ haloalkyl or C₃-C₇cycloalkyl;

each R²² is independently selected from halo, C₁-C₆ alkyl, C₂-C₆alkenyl, C₁-C₆ haloalkyl, C₁-C₆ alkoxy, C₁-C₆ haloalkoxy, phenyl,hydroxy C₁-C₆ alkyl, C₃-C₆ cycloalkyl, C₁-C₆ alkylcarbonyl, C₃-C₆cycloalkylcarbonyl, carboxy C₁-C₆ alkyl, hydroxy, amino CN, NO₂ andtrimethylsilyl, or any two R²² groups attached to adjacent ring carbonatoms can combine to form —O—(CR²³R^(23′))₁₋₆—O—;

R²³ and R^(23′) are independently H or C₁-C₃ alkyl;

or a pharmaceutically acceptable salt and/or solvate thereof.

The compounds of Formula (I) may optionally be provided in the form of apharmaceutically acceptable salt and/or solvate. In one embodiment thecompound of the invention is provided in the form of a pharmaceuticallyacceptable salt. In a second embodiment the compound of the invention isprovided in the form of a pharmaceutically acceptable solvate. In athird embodiment the compound of the invention is provided in its freeform.

In typical embodiments of the invention, R¹ is NR⁵R^(5′), such as NH₂ orNHC(═O)C₁-C₆ alkyl.

R² is typically H.

In preferred embodiments, R¹ is NH₂ and R² is H.

In alternative embodiments, R¹ is NH₂ and R² is F.

Typically in compounds of formula (I), the moiety—NHC(R¹⁵)(R^(15′))—C(═O)OR¹⁶ forms an amino acid ester residue,including natural and non-natural amino acid residues. Of particularinterest are amino acid residues wherein R^(15′) is hydrogen and R¹⁵ ismethyl, isopropyl, isobutyl or benzyl. In a typical configuration,R^(15′) is H and R¹⁵ is C₁-C₃ alkyl, such as methyl, ethyl, propyl,isopropyl.

In compounds wherein R^(15′) is hydrogen and R¹⁵ is other than hydrogen,the configuration at the asymmetric carbon atom is typically that of anL-amino acid, thus providing compounds having the stereochemistryindicated in formula (Ia):

In a preferred configuration of compounds of formula Ia, R¹⁵ is methyl.

In a further configuration of compounds of formula Ia, R¹⁵ is benzyl.

In a representative configuration of compounds of formula Ia,

R¹ is NH₂;

R² is H;

R¹³ is phenyl naphthyl or indolyl, any of which is optionallysubstituted with halo e.g. bromo or C₃-C₄ cycloalkyl e.g. cyclopropyl;

R¹⁵ is C₁-C₃ alkyl

R¹⁶ is C₁-C₈ alkyl

In a further representative configuration of compounds of formula Ia,

R¹ is NH₂;

R² is H;

R¹³ is naphthyl;

R¹⁵ is C₁-C₃ alkyl;

R¹⁶ is C₁-C₈ alkyl or benzyl;

In a further representative configuration of compounds of formula Ia,

R¹ is NH₂;

R² is H;

R¹³ is phenyl which is optionally substituted in the 4-position withhalo e.g. bromo or with C₃-C₄ cycloalkyl, e.g. cyclopropyl;

R¹⁵ is methyl;

R¹⁶ is C₃-C₈alkyl.

In a further representative configuration of compounds of formula Ia,

R¹ is NH₂;

R² is H;

R¹³ is phenyl;

R¹⁵ is methyl;

R¹⁶ is C₃-C₈ alkyl

In a further representative configuration of compounds of formula Ia,

R¹ is NH₂;

R² is F;

R¹³ is phenyl naphthyl or indolyl, any of which is optionallysubstituted with halo e.g. bromo or C₃-C₄ cycloalkyl e.g. cyclopropyl;

R¹⁵ is C₁-C₃ alkyl

R¹⁶ is C₁-C₈ alkyl

In a further representative configuration of compounds of formula Ia,

R¹ is NH₂;

R² is F;

R¹³ is naphthyl;

R¹⁵ is C₁-C₃ alkyl;

R¹⁶ is C₁-C₈ alkyl or benzyl;

In a further representative configuration of compounds of formula Ia,

R¹ is NH₂;

R² is F;

R¹³ is phenyl which is optionally substituted in the 4-position withhalo e.g. bromo or with C₃-C₄ cycloalkyl, e.g. cyclopropyl;

R¹⁵ is methyl;

R¹⁶ is C₃-C₈alkyl.

In a further representative configuration of compounds of formula Ia,

R¹ is NH₂;

R² is F;

R¹³ is phenyl;

R¹⁵ is methyl;

R¹⁶ is C₃-C₈ alkyl

In a further configuration, R¹⁵ and R^(15′) together with the carbonatom to which they are attached form C₃-C₇ cycloalkyl, for examplecyclopropyl or cyclobutyl.

R¹⁶ is typically C₁-C₁₀ alkyl or C₃-C₇ cycloalkyl.

Representative values for R¹⁶ include C₁-C₃ alkyl, such as methyl,ethyl, propyl, isopropyl. A preferred value for R¹⁶ is methyl, a furtherpreferred value for R¹⁶ is isopropyl.

In one embodiment, R¹⁶ is C₃-C₁₀ alkyl.

Representative values for R¹⁶ according to this embodiment includebranched C₅-C₈ alkyl. In one embodiment, the branching point of R¹⁶ isat C₁. In an alternative embodiment, the branching point of R¹⁶ is atC₂. Typically according to these embodiments, R^(15′) is H, and thestereochemistry at the carbon atom to which R¹⁵ is attached is that ofan L-amino acid, thus providing compounds of the general formulae:

wherein R¹⁶¹ and R¹⁶² are the same or different C₁-C₃ alkyl, and R¹⁶³and R¹⁶⁴ are the same or different C₁-C₃ alkyl.

Typically in compounds of formula (Ia′), R¹⁶ is 2-pentyl, i.e. R¹⁶¹ ispropyl and R¹⁶² is methyl.

In a further typical configuration of compounds of formula (Ia′), R¹⁶ is2-butyl, i.e. R¹⁶¹ is ethyl and R¹⁶² is methyl.

Typically in compounds of formula (Ia″), R¹⁶ is 2-propylpentyl or2-ethylbutyl, i.e. R¹⁶³ and R¹⁶⁴ are both propyl or ethyl respectively.

Further representative values for R¹⁶ include C₃-C₇cycloalkyl, such ascyclohexyl.

A further representative value for R¹⁶ is cyclopentyl.

A further representative value for R¹⁶ is benzyl.

R¹³ is typically phenyl, naphthyl or indolyl, any of which is optionallysubstituted with 1 or 2 R²².

In one embodiment of the invention, R¹³ is phenyl or naphthyl any ofwhich is optionally substituted.

In one embodiment of the invention, R¹³ is naphthyl.

In a preferred embodiment of the invention, R¹³ is phenyl.

Representative examples of R¹³ include phenyl which is optionallysubstituted with one, two or three R²², thus providing compounds of theformula (II-aa):

wherein each R²², when present, is independently selected from halo,C₁-C₆ alkyl, C₂-C₆ alkenyl and C₁-C₆ alkoxy. Typically, the phenyl ringis unsubstituted or substituted with one R²².

In one configuration of compounds of Formula (II-aa), the phenyl ring isunsubstituted.

In a further configuration of compounds of Formula (II-aa), the phenylring is substituted with one R²². Typically in this configuration, thesubstituent R²² is located to the 4-position of the phenyl ring.

In one embodiment of compounds of the inventions, R¹³ is phenyl which issubstituted in the 4-position with halo, e.g. bromo or with C₃-C₄cycloalkyl, e.g. cyclopropyl.

In one configuration of compounds of Formula (II-aa), the phenyl ring issubstituted with carboxy C₁-C₆ alkyl. A representative example of thisconfiguration is illustrated in the partial formula:

In a further configuration of compounds of Formula (II-aa), the phenylring is substituted with two R²² located on adjacent carbon atoms andthe two R²² combine to form —O—CH₂—O—, thus forming the partialstructure:

Further representative values for R¹³ include optionally substitutedpyridyl. Typically, the pyridyl moiety is unsubstituted or substitutedwith one or two substituents each independently selected from halo,C₁-C₆ haloalkyl, C₁-C₆ alkyl, C₂-C₆ alkenyl, C₁-C₆ alkoxy, hydroxy,amino.

In a typical embodiment of compounds of formula (I),

R¹ is NH₂ or NHC(═O)C₁-C₆ alkyl;

R¹³ is phenyl, naphthyl or indolyl, any of which is optionallysubstituted with halo, C₁-C₃ alkyl, C₁-C₃ alkoxy, C₃-C₆ cycloalkyl orC₁-C₃ haloalkyl;

R^(15′) is H and R¹⁵ is C₁-C₃ alkyl or benzyl;

R¹⁶ is C₁-C₁₀ alkyl or C₃-C₇ cycloalkyl.

In a typical embodiment of compounds of formula (I) or (Ia),

R¹ is NH₂ or NHC(═O)C₁-C₆ alkyl;

R¹³ is phenyl or naphthyl, any of which is optionally substituted withhalo, C₁-C₃ alkyl, C₁-C₃ alkoxy, C₃-C₆ cycloalkyl or C₁-C₃ haloalkyl;

R^(15′) is H and R¹⁵ is C₁-C₃ alkyl or benzyl;

R¹⁶ is C₂-C₁₀ alkyl or C₃-C₇ cycloalkyl.

In a further typical embodiment of compounds of formula (I),

R¹ is NH₂;

R² is H;

R¹³ is phenyl;

R^(15′) is H and R¹⁵ is C₁-C₃ alkyl;

R¹⁶ is C₁-C₃ alkyl or cyclohexyl.

In a further typical embodiment of compounds of Formula (I) or (Ia),

R¹ is NH₂;

R² is H;

R¹³ is phenyl;

R^(15′) is H and R¹⁵ is C₁-C₃ alkyl or benzyl;

R¹⁶ is C₃-C₈ alkyl, cyclopentyl or cyclohexyl.

The compounds of the present invention show activity against cancerespecially liver cancer such as HCC, and can be used as medicine in thetreatment of warm-blooded animals, particularly humans, having cancer.Especially the compounds can be used as medicine in the treatment ofhumans having liver cancer such as HCC.

In order to avoid undesired side effects, particularly toxicity in otherorgans, delivery of the drug to the site of the tumour while reducingexposure to normal tissue is crucial. The compounds of the invention arestable in gastric fluid but readily metabolized by liver enzymes, theymay therefore be absorbed in the stomach and transported as a maskedcytotoxic agent to the liver where absorption, metabolism and formationof the active cytotoxic triphosphate occurs. Accordingly, the inventionprovides compounds which are absorbed and processed primarily in theliver, thus minimizing exposure to other organs in the body and toxicside effects.

Without wishing to be bound by theory, the anti-oncogenic activity ofthe compounds of the invention may be exerted directly against cellularprocesses of the rapidly acting tumourogenic cells of the cancer, butmay additionally or alternatively exert their effects through modulationof the tumour's microenvironment, such as inhibition of angiogenesis,thereby starving the tumour of nourishment leading to inhibition oftumour growth.

The compounds of the present invention are also useful in the treatmentof secondary liver cancers, metastasis in the liver, i.e. cancer thatoriginate from organs elsewhere in the body, such as the colon, lung orbreast and migrates to the liver.

The present invention also relates to a method of treating warm-bloodedanimals, in particular humans, having cancer, especially liver cancersuch as HCC, said method comprises the administration of an effectiveamount of a compound of Formula (I) or any subgroup thereof.

The present invention also relates to a method of treating warm-bloodedanimals, in particular humans, having a secondary liver cancer, saidmethod comprises the administration of an effective amount of a compoundof Formula (I) or any subgroup thereof.

Said use as a medicine or method of treatment comprises the systemicadministration to a subject having cancer of an effective amount of acompound of Formula (I).

In one aspect, the invention provides a pharmaceutical compositioncomprising a compound of Formula (I) in association with apharmaceutically acceptable adjuvant, diluent, excipient or carrier.

In a further aspect, the invention provides a pharmaceutical compositionfor use in the treatment of cancer comprising a compound of Formula (I)in association with a pharmaceutically acceptable adjuvant, diluent,excipient or carrier.

In a further aspect, the invention provides a pharmaceutical compositionfor use in the treatment of liver cancer, such as HCC comprising acompound of Formula (I) in association with a pharmaceuticallyacceptable adjuvant, diluent, excipient or carrier.

In a further aspect, the invention provides a pharmaceutical compositionfor use in the treatment of a secondary liver cancer comprising acompound of Formula (I) in association with a pharmaceuticallyacceptable adjuvant, diluent, excipient or carrier.

In a further aspect, the invention relates to a process of preparing apharmaceutical composition as specified herein, which comprisesintimately mixing a pharmaceutically acceptable adjuvant, diluent,excipient and/or carrier with a therapeutically effective amount of acompound of Formula (I).

In a further aspect, the invention provides a pharmaceutical compositionfor use in the treatment or inhibition mentioned above, which furthercomprises one or more additional therapeutic agents.

The pharmaceutical compositions mentioned above will typically containan effective amount (e.g. for humans) of the compound of Formula (I),although sub-therapeutic amounts of the compound of Formula (I) maynevertheless be of value when intended for use in combination with otheragents or in multiple doses.

In this context a therapeutically effective amount is an amountsufficient to produce an intended result. The therapeutically effectiveamount will vary depending on individual requirements in each particularcase. Features that influence the dose are e.g. the severity of thedisease to be treated, age, weight, general health condition etc. of thesubject to be treated. With respect to an anti-cancer effect, thateffect may be inhibition of further tumour growth, reduction of thelikelihood or elimination of metastasis or producing cell death in thetumour, resulting in a shrinkage of the tumour or preventing theregrowth of a tumour after the patient's tumour is in remission.

In a further aspect, the present invention provides a compound ofFormula (I) for use as a medicament.

In a further aspect, the present invention provides a compound ofFormula (I) for use in the treatment of cancer.

In a further aspect, the present invention provides a compound ofFormula (I) for use in the treatment of liver cancer such as HCC.

In a further aspect, the present invention provides a compound ofFormula (I) for use in the treatment of a secondary liver cancer.

In a further aspect, the present invention provides a compound ofFormula (I) for use in the treatment as described above in combinationwith one or more additional cancer treatment(s) such as otheranti-cancer drugs, surgery, immunotherapy and/or regional therapies likeradiofrequency ablation.

In a further embodiment, an additional anticancer treatment isradiotherapy.

In one embodiment, an additional anticancer treatment is one or moreother nucleoside analogue(s) which exhibit potent antitumor activity.

In one aspect the present invention provides a pharmaceuticalcombination comprising a therapeutically effective amount of compound offormula and one or more additional therapeutic agent(s) selected fromthe group consisting of chemotherapeutical agent, multi-drug resistancereversing agent and biological response modifier.

In one embodiment of this aspect, a further therapeutic agent is achemotherapeutical agent.

In a further aspect, the present invention provides a compound ofFormula (I) for use in the manufacture of a medicament.

In a further aspect, the present invention provides a compound ofFormula (I) for use in the manufacture of a medicament for the treatmentof cancer.

In a further aspect, the present invention provides a compound ofFormula (I) for use in the manufacture of a medicament for the treatmentof liver cancer such as HCC.

In a further aspect, the present invention provides a compound ofFormula (I) for use in the manufacture of a medicament for the treatmentof a secondary liver cancer.

In a further aspect, the present invention provides a method for thetreatment of cancer comprising the administration of a therapeuticallyeffective amount of a compound of Formula (I), to a subject, e.g. ahuman in need thereof.

In a further aspect, the present invention provides a method for thetreatment of liver cancer such as HCC, comprising the administration ofa therapeutically effective amount of a compound of Formula (I) to asubject, e.g. a human in need thereof.

In a further aspect, the present invention provides a method for thetreatment of a secondary liver cancer, comprising the administration ofa therapeutically effective amount of a compound of Formula (I) to asubject, e.g. a human in need thereof.

In a further aspect, the present invention provides a method for thetreatment as described above in combination with additional cancertreatment(s) such as other anti-cancer drugs, surgery, immunotherapyand/or regional therapies like radiofrequency ablation.

In one aspect the present invention provides a method of treatment of aprimary or secondary liver cancer comprising the administration of apharmaceutical combination comprising a therapeutically effective amountof compound of formula I, further comprising one or more additionaltherapeutic agent(s) selected from the group consisting ofchemotherapeutical agent, multi-drug resistance reversing agent andbiological response modifier.

In one embodiment of this aspect, a further therapeutic agent is achemotherapeutic agent. In one aspect, the invention provides a compoundof Formula (I) which is selected from the compounds depicted below:

or a pharmaceutically acceptable salt thereof.

Furthermore, the invention relates to a method for manufacturing acompound of Formula (I), to novel intermediates for use in themanufacture of compounds of Formula (I) and to the manufacture of suchintermediates.

Whenever the term ‘compounds of Formula (I)’, ‘the compounds of theinvention”, “the compounds of the present invention” or similar terms isused in the foregoing and hereinafter, it is meant to include thecompounds of Formula (I) and any subgroup of compounds of Formula (I),including all possible stereochemically isomeric forms, theirpharmaceutically acceptable salts, solvates, quaternary amines and metalcomplexes.

The compounds of the present invention may be formulated into variouspharmaceutical forms for administration purposes. As appropriatecompositions there may be cited all compositions usually employed fororal administration of drugs. To prepare the pharmaceutical compositionsof this invention, an effective amount of the particular compound,optionally in addition salt form or solvate, as the active ingredient iscombined in intimate admixture with a pharmaceutically acceptablecarrier, which carrier may take a wide variety of forms depending on theform of preparation desired for administration. These pharmaceuticalcompositions are desirable in unitary dosage form suitable for oraladministration. For example, in preparing the compositions in oraldosage form, any of the usual pharmaceutical media may be employed suchas, for example, water, glycols, oils, alcohols and the like in the caseof oral liquid preparations such as suspensions, syrups, elixirs,emulsions and solutions; or solid carriers such as starches, sugars,kaolin, lubricants, binders, disintegrating agents and the like in thecase of powders, pills, capsules, and tablets. Because of their ease inadministration, tablets and capsules represent the most advantageousoral dosage unit forms, in which case solid pharmaceutical carriers areobviously employed. Also included are solid form preparations intendedto be converted, shortly before use, to liquid form preparations.

It is especially advantageous to formulate the aforementionedpharmaceutical compositions in unit dosage form for ease ofadministration and uniformity of dosage. Unit dosage form as used hereinrefers to physically discrete units suitable as unitary dosages, eachunit containing a predetermined quantity of active ingredient calculatedto produce the desired therapeutic effect in association with therequired pharmaceutical carrier. Examples of such unit dosage forms aretablets (including scored or coated tablets), capsules, pills, powderpackets, wafers and the like, and segregated multiples thereof.

In general it is contemplated that an an-cancer effective daily amountwould be from about 0.01 to about 700 mg/kg, or about 0.5 to about 400mg/kg, or about 1 to about 250 mg/kg, or about 2 to about 200 mg/kg, orabout 10 to about 150 mg/kg body weight. It may be appropriate toadminister the required dose as two, three, four or more sub-doses atappropriate intervals throughout the day. Said sub-doses may beformulated as unit dosage forms, for example, containing about 1 toabout 5000 mg, or about 50 to about 3000 mg, or about 100 to about 1000mg, or about 200 to about 600 mg, or about 100 to about 400 mg of activeingredient per unit dosage form.

The compounds of the present invention may exhibit an anticancer effectalone and/or enhance the ability of another anti-cancer agent to exhibitan anticancer effect.

The compounds of the invention are represented as a definedstereoisomer. The absolute configuration of such compounds can bedetermined using art-known methods such as, for example, X-raydiffraction or NMR and/or implication from start materials of knownstereochemistry. Pharmaceutical compositions in accordance with theinvention will preferably comprise substantially stereoisomerically purepreparations of the indicated stereoisomer.

Pure stereoisomeric forms of the compounds and intermediates asmentioned herein are defined as isomers substantially free of otherenantiomeric or diastereomeric forms of the same basic molecularstructure of said compounds or intermediates. In particular, the term“stereoisomerically pure” concerns compounds or intermediates having astereoisomeric excess of at least 80% (i.e. minimum 90% of one isomerand maximum 10% of the other possible isomers) up to a stereoisomericexcess of 100% (i.e. 100% of one isomer and none of the other), more inparticular, compounds or intermediates having a stereoisomeric excess of90% up to 100%, even more in particular having a stereoisomeric excessof 94% up to 100% and most in particular having a stereoisomeric excessof 97% up to 100%. The terms “enantiomerically pure” and“diastereomerically pure” should be understood in a similar way, butthen having regard to the enantiomeric excess, and the diastereomericexcess, respectively, of the mixture in question.

Pure stereoisomeric forms of the compounds and intermediates of thisinvention may be obtained by the application of art-known procedures.For instance, enantiomers may be separated from each other by theselective crystallization of their diastereomeric salts with opticallyactive acids or bases. Examples thereof are tartaric acid,dibenzoyltartaric acid, ditoluoyltartaric acid and camphorsulfonic acid.Alternatively, enantiomers may be separated by chromatographictechniques using chiral stationary phases. Said pure stereochemicallyisomeric forms may also be derived from the corresponding purestereochemically isomeric forms of the appropriate starting materials,provided that the reaction occurs stereospecifically.

Preferably, if a specific stereoisomer is desired, said compound issynthesized by stereospecific methods of preparation. These methods willadvantageously employ enantiomerically pure starting materials.

The diastereomeric racemates of the compounds of the invention can beobtained separately by conventional methods. Appropriate physicalseparation methods that may advantageously be employed are, for example,selective crystallization and chromatography, e.g. columnchromatography.

When a phosphorus atom is present in a compound of the invention, thephosphorus atom may represent a chiral centre. The chirality at thiscentre is designated “R” or “S” according to the Cahn-Ingold-Prelogpriority rules. When the chirality is not indicated, it is contemplatedthat both the R- and S-isomers are meant to be included, as well as amixture of both, i.e. a diastereomeric mixture.

In preferred embodiments of the invention, the stereoisomers having theS-configuration at the phosphorus atom are included. These stereoisomersare designated S_(P).

In other embodiments of the invention, the stereoisomers having theR-configuration at the phosphorus atom are included. These stereoisomersare designated R_(P).

In other embodiments of the invention, diastereomeric mixtures areincluded, i.e. mixtures of compounds having the R- or S-configuration atthe phosphorus atom.

The present invention also includes isotope-labelled compounds ofFormula (I), wherein one or more of the atoms is replaced by an isotopeof that atom, i.e. an atom having the same atomic number as, but anatomic mass different from, the one(s) typically found in nature.Examples of isotopes that may be incorporated into the compounds ofFormula (I), include but are not limited to isotopes of hydrogen, suchas ²H and ³H (also denoted D for deuterium and T for tritium,respectively), carbon, such as ¹¹C, ¹³C and ¹⁴C, nitrogen, such as ¹³Nand ¹⁵N, oxygen, such as ¹⁵O, ¹⁷O and ¹⁸O, phosphorus, such as ³¹P and³²P, sulfur, such as ³⁵S, fluorine, such as ¹⁸F, chlorine, such as ³⁶Cl,bromine such as ⁷⁵Br, ⁷⁶Br, ⁷⁷Br and ⁸²Br, and iodine, such as ¹²³I,¹²⁴I, ¹²⁵I and ¹³¹I. The choice of isotope included in anisotope-labelled compound will depend on the specific application ofthat compound. For example, for drug or substrate tissue distributionassays, compounds wherein a radioactive isotope such as ³H or ¹⁴C isincorporated will generally be most useful. For radio-imagingapplications, for example positron emission tomography (PET) a positronemitting isotope such as ¹¹C, ¹⁸F, ¹³N or ¹⁵O will be useful. Theincorporation of a heavier isotope, such as deuterium, i.e. ²H, mayprovide greater metabolic stability to a compound of Formula (I) whichmay result in, for example, an increased in vivo half life of thecompound or reduced dosage requirements.

Isotope-labelled compounds of the invention can be prepared by processesanalogous to those described in the Schemes and/or Examples herein belowby using the appropriate isotope-labelled reagent or starting materialinstead of the corresponding non-isotope-labelled reagent or startingmaterial, or by conventional techniques known to those skilled in theart.

The pharmaceutically acceptable addition salts comprise thetherapeutically active acid and base addition salt forms of thecompounds of Formula (I). Of interest are the free, i.e. non-salt formsof the compounds of Formula (I) or any subgroup thereof.

The pharmaceutically acceptable acid addition salts can conveniently beobtained by treating the base form with such appropriate acid.Appropriate acids comprise, for example, inorganic acids such ashydrohalic acids, e.g. hydrochloric or hydrobromic acid, sulfuric,nitric, phosphoric and the like acids; or organic acids such as, forexample, acetic, propionic, hydroxyacetic, lactic, pyruvic, oxalic (i.e.ethanedioic), malonic, succinic (i.e. butanedioic acid), maleic,fumaric, malic (i.e. hydroxylbutanedioic acid), tartaric, citric,methanesulfonic, ethanesulfonic, benzenesulfonic, p-toluenesulfonic,cyclamic, salicylic, p-aminosalicylic, pamoic and the like acids.Conversely said salt forms can be converted by treatment with anappropriate base into the free base form.

The compounds of Formula (I) containing an acidic proton may also beconverted into their non-toxic metal or amine addition salt forms bytreatment with appropriate organic and inorganic bases. Appropriate basesalt forms comprise, for example, the ammonium salts, the alkali andearth alkaline metal salts, e.g. the lithium, sodium, potassium,magnesium, calcium salts and the like, salts with organic bases, e.g.the benzathine, N-methyl-D-glucamine, hydrabamine salts, and salts withamino acids such as, for example, arginine, lysine and the like.

Some of the compounds of Formula (I) may also exist in their tautomericform. For example, tautomeric forms of amide groups (—C(═O)—NH—) areiminoalcohols (—C(OH)═N—), which can become stabilized in rings witharomatic character. Such forms, although not explicitly indicated in thestructural formulae represented herein, are intended to be includedwithin the scope of the present invention.

The terms and expressions used herein throughout the abstract,specification and claims shall be interpreted as defined below unlessotherwise indicated. The meaning of each term is independent at eachoccurrence. These definitions apply regardless of whether a term is usedby itself or in combination with other terms, unless otherwiseindicated. A term or expression used herein which is not explicitlydefined, shall be interpreted as having its ordinary meaning used in theart. Chemical names, common names, and chemical structures may be usedinterchangeably to describe the same structure. If a chemical compoundis referred to using both a chemical structure and a chemical name andan ambiguity exists between the structure and the name, the structurepredominates.

“C_(m)-C_(n)alkyl” on its own or in composite expressions such asC_(m)-C_(n)haloalkyl, C_(m)-C_(n)alkylcarbonyl, C_(m)-C_(n)alkylamine,etc. represents a straight or branched aliphatic hydrocarbon radicalhaving the number of carbon atoms designated, e.g. C₁-C₄ alkyl means analkyl radical having from 1 to 4 carbon atoms. C₁-C₆ alkyl has acorresponding meaning, including also all straight and branched chainisomers of pentyl and hexyl. Preferred alkyl radicals for use in thepresent invention are C₁-C₆ alkyl, including methyl, ethyl, n-propyl,isopropyl, n-butyl, isobutyl, sec-buty, tert-butyl, n-pentyl andn-hexyl, especially C₁-C₄ alkyl such as methyl, ethyl, n-propyl,isopropyl, t-butyl, n-butyl and isobutyl. Methyl and isopropyl aretypically preferred. An alkyl group may be unsubstituted or substitutedby one or more substituents which may be the same or different, eachsubstituent being independently selected from the group consisting ofhalo, alkenyl, alkynyl, aryl, cycloalkyl, cyano, hydroxy, —O-alkyl,—O-aryl, -alkylene-O-alkyl, alkylthio, —NH₂, —NH(alkyl), —N(alkyl)₂,—NH(cycloalkyl), —O—C(═O)-alkyl, —O—C(═O)-aryl, —O—C(═O)-cycloalkyl,—C(═O)OH and —C(═O)O-alkyl. It is generally preferred that the alkylgroup is unsubstituted, unless otherwise indicated.

“C₂-C_(n)alkenyl” represents a straight or branched aliphatichydrocarbon radical containing at least one carbon-carbon double bondand having the number of carbon atoms designated, e.g. C₂-C₄ alkenylmeans an alkenyl radical having from 2 to 4 carbon atoms; C₂-C₆ alkenylmeans an alkenyl radical having from 2 to 6 carbon atoms. Non-limitingalkenyl groups include ethenyl, propenyl, n-butenyl, 3-methylbut-2-enyl,n-pentenyl and hexenyl. An alkenyl group may be unsubstituted orsubstituted by one or more substituents which may be the same ordifferent, each substituent being independently selected from the groupconsisting of halo, alkenyl, alkynyl, aryl, cycloalkyl, cyano, hydroxy,—O-alkyl, —O-aryl, -alkylene-O-alkyl, alkylthio, —NH₂, —NH(alkyl),—N(alkyl)₂, —NH(cycloalkyl), —O—C(═O)-alkyl, —O—C(═O)-aryl,—O—C(═O)-cycloalkyl, —C(═O)OH and —C(═O)O-alkyl. It is generallypreferred that the alkenyl group is unsubstituted, unless otherwiseindicated.

“C₂-C_(n)alkynyl” represents a straight or branched aliphatichydrocarbon radical containing at least one carbon-carbon triple bondand having the number of carbon atoms designated, e.g. C₂-C₄ alkynylmeans an alkynyl radical having from 2 to 4 carbon atoms; C₂-C₆ alkynylmeans an alkynyl radical having from 2 to 6 carbon atoms. Non-limitingalkenyl groups include ethynyl, propynyl, 2-butynyl and 3-methylbutynylpentynyl and hexynyl. An alkynyl group may be unsubstituted orsubstituted by one or more substituents which may be the same ordifferent, each substituent being independently selected from the groupconsisting of halo, alkenyl, alkynyl, aryl, cycloalkyl, cyano, hydroxy,—O-alkyl, —O-aryl, -alkylene-O-alkyl, alkylthio, —NH₂, —NH(alkyl),—N(alkyl)₂, —NH(cycloalkyl), —O—C(═O)-alkyl, —O—C(═O)-aryl,—O—C(═O)-cycloalkyl, —C(O)OH and —C(O)O-alkyl. It is generally preferredthat the alkynyl group is unsubstituted, unless otherwise indicated.

The term “C_(m)-C_(n)haloalkyl” as used herein representsC_(m)-C_(n)alkyl wherein at least one C atom is substituted with ahalogen (e.g. the C_(m)-C_(n)haloalkyl group may contain one to threehalogen atoms), preferably chloro or fluoro. Typical haloalkyl groupsare C_(m)-C_(n)haloalkyl, in which halo suitably represents fluoro.Exemplary haloalkyl groups include fluoromethyl, difluromethyl andtrifluoromethyl.

The term “C_(m)-C_(n)hydroxyalkyl” as used herein representsC_(m)-C_(n)alkyl wherein at least one C atom is substituted with onehydroxy group. Typical C_(m)-C_(n)hydroxyalkyl groups areC_(m)-C_(n)alkyl wherein one C atom is substituted with one hydroxygroup. Exemplary hydroxyalkyl groups include hydroxymethyl andhydroxyethyl.

The term “C_(m)-C_(n)aminoalkyl” as used herein representsC_(m)-C_(n)alkyl wherein at least one C atom is substituted with oneamino group. Typical C_(m)-C_(n)aminoalkyl groups are C_(m)-C_(n)alkylwherein one C atom is substituted with one amino group. Exemplaryaminoalkyl groups include aminomethyl and aminoethyl.

The term “C_(m)-C_(n)alkylene” as used herein represents a straight orbranched bivalent alkyl radical having the number of carbon atomsindicated. Preferred C_(m)-C_(n)alkylene radicals for use in the presentinvention are C₁-C₃ alkylene. Non-limiting examples of alkylene groupsinclude —CH₂—, —CH₂CH₂—, —CH₂CH₂CH₂—, —CH(CH₃)CH₂CH₂—, —CH(CH₃)— and—CH(CH(CH₃)₂)—.

The term “Me” means methyl, and “MeO” means methoxy.

The term “C_(m)-C_(n)alkylcarbonyl” represents a radical of the formulaC_(m)-C_(n)alkyl-C(═O)— wherein the C_(m)-C_(n)alkyl moiety is asdefined above. Typically, “C_(m)-C_(n)alkylcarbonyl” isC₁-C₆alkyl-C(═O)—.

“C_(m)-C_(n)alkoxy” represents a radical C_(m)-C_(n)alkyl-O— whereinC_(m)-C_(n)alkyl is as defined above. Of particular interest is C₁-C₄alkoxy which includes methoxy, ethoxy, n-propoxy, isopropoxy, t-butoxy,n-butoxy and isobutoxy. Methoxy and isopropoxy are typically preferred.C₁-C₆ alkoxy has a corresponding meaning, expanded to include allstraight and branched chain isomers of pentoxy and hexoxy.

The term “C_(m)-C_(n)alkoxycarbonyl” represents a radical of the formulaC_(m)-C_(n)alkoxy-C(═O)— wherein the C_(m)-C_(n)alkoxy moiety is asdefined above. Typically, “C_(m)-C_(n)alkoxycarbonyl” is C₁-C₆alkoxy-C(═O)—.

The term “amino” represents the radical —NH₂.

The term “halo” represents a halogen radical such as fluoro, chloro,bromo or iodo. Typically, halo groups are fluoro or chloro.

The term “aryl” means a phenyl, biphenyl or naphthyl group.

The term “heterocycloalkyl” represents a stable saturated monocyclic 3-7membered ring containing 1-3 heteroatoms independently selected from O,S and N. In one embodiment the stable saturated monocyclic 3-7 memberedring contains 1 heteroatom selected from O, S and N. In a secondembodiment the stable saturated monocyclic 3-7 membered ring contains 2heteroatoms independently selected from O, S and N. In a thirdembodiment the stable saturated monocyclic 3-7 membered ring contains 3heteroatoms independently selected from O, S and N. The stable saturatedmonocyclic 3-7 membered ring containing 1-3 heteroatoms independentlyselected from O, S and N may typically be a 5-7 membered ring, such as a5 or 6 membered ring. A heterocycloalkyl group may be unsubstituted orsubstituted by one or more substituents which may be the same ordifferent, each substituent being independently selected from the groupconsisting of halo, alkenyl, alkynyl, aryl, cycloalkyl, cyano, hydroxy,—O-alkyl, —O-aryl, -alkylene-O-alkyl, alkylthio, —NH₂, —NH(alkyl),—N(alkyl)₂, —NH(cycloalkyl), —O—C(═O)-alkyl, —O—C(═O)-aryl,—O—C(═O)-cycloalkyl, —C(═O)OH and —C(═O)O-alkyl. It is generallypreferred that the heterocycloalkyl group is unsubstituted, unlessotherwise indicated.

The term “heteroaryl” represents a stable mono or bicyclic aromatic ringsystem containing 1-4 heteroatoms independently selected from O, S andN, each ring having 5 or 6 ring atoms. In one embodiment of theinvention the stable mono or bicyclic aromatic ring system contains oneheteroatom selected from O, S and N, each ring having 5 or 6 ring atoms.In a second embodiment of the invention the stable mono or bicyclicaromatic ring system contains two heteroatoms independently selectedfrom O, S and N, each ring having 5 or 6 ring atoms. In a thirdembodiment the stable mono or bicyclic aromatic ring system containsthree heteroatoms independently selected from O, S and N, each ringhaving 5 or 6 ring atoms. In a fourth embodiment the stable mono orbicyclic aromatic ring system contains four heteroatoms independentlyselected from O, S and N, each ring having 5 or 6 ring atoms.

One embodiment of heteroaryl comprises flavone.

The term “C₃-C_(n)cycloalkyl” represents a cyclic monovalent alkylradical having the number of carbon atoms indicated, e.g. C₃-C₇cycloalkyl means a cyclic monovalent alkyl radical having from 3 to 7carbon atoms. Preferred cycloalkyl radicals for use in the presentinvention are C₃-C₄ alkyl i.e. cyclopropyl and cyclobutyl. A cycloalkylgroup may be unsubstituted or substituted by one or more substituentswhich may be the same or different, each substituent being independentlyselected from the group consisting of halo, alkenyl, alkynyl, aryl,cycloalkyl, cyano, hydroxy, —O-alkyl, —O-aryl, -alkylene-O-alkyl,alkylthio, —NH₂, —NH(alkyl), —N(alkyl)₂, —NH(cycloalkyl),—O—C(═O)-alkyl, —O—C(═O)-aryl, —O—C(═O)-cycloalkyl, —C(═O)OH and—C(═O)O-alkyl. It is generally preferred that the cycloalkyl group isunsubstituted, unless otherwise indicated.

The term “amino C_(m)-C_(n)alkyl” represents a C_(m)-C_(n)alkyl radicalas defined above which is substituted with an amino group, i.e. onehydrogen atom of the alkyl moiety is replaced by an NH₂-group.Typically, “amino C_(m)-C_(n)alkyl” is amino C₁-C₆ alkyl.

The term “amino C_(m)-C_(n)alkylcarbonyl” represents aC_(m)-C_(n)alkylcarbonyl radical as defined above, wherein one hydrogenatom of the alkyl moiety is replaced by an NH₂-group. Typically, “aminoC_(m)-C_(n)alkylcarbonyl” is amino C₁-C₆alkylcarbonyl. Examples of aminoC_(m)-C_(n)alkylcarbonyl include but are not limited to glycyl:C(═O)CH₂NH₂, alanyl: C(═O)CH(NH₂)CH₃, valinyl: C═OCH(NH₂)CH(CH₃)₂,leucinyl: C(═O)CH(NH₂)(CH₂)₃CH₃, isoleucinyl:C(═O)CH(NH₂)CH(CH₃)(CH₂CH₃) and norleucinyl: C(═O)CH(NH₂)(CH₂)₃CH₃ andthe like. This definition is not limited to naturally occurring aminoacids.

As used herein, the term “(═O)” forms a carbonyl moiety when attached toa carbon atom. It should be noted that an atom can only carry an oxogroup when the valency of that atom so permits.

The term “monophosphate, diphosphate and triphosphate ester” refers tothe groups:

As used herein, the radical positions on any molecular moiety used inthe definitions may be anywhere on such a moiety as long as it ischemically stable. When any variable present occurs more than once inany moiety, each definition is independent.

The term “solvates” covers any pharmaceutically acceptable solvates thatthe compounds of Formula (I) as well as the salts thereof, are able toform. Such solvates are for example hydrates, alcoholates, e.g.ethanolates, propanolates, and the like, especially hydrates.

The term “prodrug” as used herein denotes a compound that is a drugprecursor which upon administration to a subject are readily convertiblein vivo by metabolic and/or chemical processes to yield the activecompound.

The expression “liver targeted prodrug” as used herein denotes a prodrugwhich is metabolised to its active species predominantly in the liver.

The expression “liver cancer” as used herein is meant to include bothprimary and secondary liver cancer, i.e. cancer that origins in theliver, and liver metastasis from cancer in other organs respectively.

Related terms are to be interpreted in line with the definitionsprovided above and the common usage in the technical field.

In general, the names of compounds used in this application aregenerated using ChemDraw Ultra 12.0. In addition, if the stereochemistryof a structure or a portion of a structure is not indicated with forexample bold or dashed lines, the structure or portion of that structureis to be interpreted as encompassing all stereoisomers of it.

General Synthetic Methods

Compounds of the present invention may be prepared by a variety ofmethods e.g. as depicted in the illustrative synthetic schemes shown anddescribed below. The starting materials and reagents used are availablefrom commercial suppliers or can be prepared according to literatureprocedures set forth in references using methods well known to thoseskilled in the art.

Scheme 1 illustrates a general route to compounds of Formula (I).

Condensation of commercially available troxacitabine derivative (1a),prepared as described above, with a desired phosphoramidate reagent (1b)wherein Lg is a suitable leaving group such as a halogen like chlorideor an activated phenol like pentachlorophenol, p-nitrophenol,pentafluorophenol or the like, in an inert solvent such as an ether,e.g. diethyl ether or THF, or a halogenated hydrocarbon, e.g.dichloromethane, in the presence of a base such as a N-methylimidazole(NMI) or a Grignard reagent like tert.butylmagnesium chloride or thelike, the phosphoramidate derivative (1c).

Phosphoramidate reagents (1b) to be used in the above scheme wherein Lgis chloro, i.e. phosphoramidochloridates, can be prepared in a two-stepreaction starting from phosphorus oxychloride (POCl₃) as illustrated inScheme 2.

Condensation of POCl₃ with a desired alcohol R¹³OH in an inert solventlike Et₂O provides alkoxy or aryloxy phosphorodichloridate (2a).Subsequent reaction with an amino acid derivative (2b) provides thephosphoramidochloridates wherein R^(3′) is H (2c).

If desired, the obtained phosphoramidochloridates (2c) may be convertedto the corresponding phosphorylating agent having an activated phenol asleaving group, for instance pentaflurorophenol or p-NO₂-phenol asgenerally illustrated in Scheme 3.

This conversion is conveniently performed by reaction of the chloroderivative (2c) with the desired activated phenol in the presence of abase like triethylamine or similar, thus providing phosphorylatingagents (3a) and (3b).

The use of various protecting groups (PG) used in schemes above areknown to the skilled person, and their utility and further alternativesare extensively described in the literature, see for instance Greene T.W., Wuts P. G. M. Protective groups in organic synthesis, 2nd ed. NewYork: Wiley; 1995.

The term “N-protecting group” or “N-protected” as used herein refers tothose groups intended to protect the N-terminus of an amino acid orpeptide or to protect an amino group against undesirable reactionsduring synthetic procedures. Commonly used N-protecting groups aredisclosed in Greene. N-protecting groups include acyl groups such asformyl, acetyl, propionyl, pivaloyl, t-butylacetyl, 2-chloroacetyl,2-bromoacetyl, trifluoroacetyl, trichloroacetyl, phthalyl,o-nitrophenoxyacetyl, α-chlorobutyryl, benzoyl, 4-chlorobenzoyl,4-bromobenzoyl, 4-nitrobenzoyl, and the like; sulfonyl groups such asbenzenesulfonyl, p-toluenesulfonyl, and the like; carbamate forminggroups such as benzyloxycarbonyl, p-chlorobenzyloxy-carbonyl,p-methoxybenzyloxycarbonyl, p-nitrobenzyloxycarbonyl,2-nitrobenzyloxycarbonyl, p-bromobenzyloxycarbonyl,3,4-dimethoxybenzyloxycarbonyl, 4-methoxybenzyloxycarbonyl,2-nitro-4,5-dimethoxybenzyloxycarbonyl,3,4,5-trimethoxybenzyloxycarbonyl,1-(p-biphenyl)-1-methylethoxycarbonyl,α,α-dimethyl-3,5-dimethoxybenzyloxycarbonyl, benzhydryloxycarbonyl,t-butoxycarbonyl, diisopropylmethoxycarbonyl, isopropyloxycarbonyl,ethoxycarbonyl, methoxycarbonyl, allyloxycarbonyl,2,2,2-trichloroethoxycarbonyl, phenoxycarbonyl, 4-nitrophenoxycarbonyl,fluorenyl-9-methoxycarbonyl, cyclopentyloxycarbonyl,adamantyloxycarbonyl, cyclohexyloxycarbonyl, phenylthiocarbonyl, and thelike; alkyl groups such as benzyl, triphenylmethyl, benzyloxymethyl andthe like; and silyl groups such as trimethylsilyl and the like. FavouredN-protecting groups include formyl, acetyl, benzoyl, pivaloyl,t-butylacetyl, phenylsulfonyl, benzyl (Bz), t-butoxycarbonyl (BOC) andbenzyloxycarbonyl (Cbz).

Hydroxy and/or carboxy protecting groups are also extensively reviewedin Greene ibid and include ethers such as methyl, substituted methylethers such as methoxymethyl, methylthiomethyl, benzyloxymethyl,t-butoxymethyl, 2-methoxyethoxymethyl and the like, silyl ethers such astrimethylsilyl (TMS), t-butyldimethylsilyl (TBDMS) tribenzylsilyl,triphenylsilyl, t-butyldiphenylsilyl, triisopropyl silyl and the like,substituted ethyl ethers such as 1-ethoxymethyl,1-methyl-1-methoxyethyl, t-butyl, allyl, benzyl, p-methoxybenzyl,diphenylmethyl, triphenylmethyl and the like, aralkyl groups such astrityl, and pixyl (9-hydroxy-9-phenylxanthene derivatives, especiallythe chloride). Ester hydroxy protecting groups include esters such asformate, benzylformate, chloroacetate, methoxyacetate, phenoxyacetate,pivaloate, adamantoate, mesitoate, benzoate and the like. Carbonatehydroxy protecting groups include methyl vinyl, allyl, cinnamyl, benzyland the like.

DETAILED DESCRIPTION OF THE EMBODIMENTS

Various embodiments of the invention and intermediates therefore willnow be illustrated by the following examples. The Examples are justintended to further illustrate the invention and are by no meanslimiting the scope of the invention. The compound names were generatedby ChemDraw Ultra software, Cambridgesoft, version 12.0.2.

In addition to the definitions above, the following abbreviations areused in the synthetic schemes above and the examples below. If anabbreviation used herein is not defined it has its generally acceptedmeaning.

Bn Benzyl

BOP-Cl Bis(2-oxo-3-oxazolidinyl)phosphinic chloride

DCC Dicyclohexylcarbodiimide

DCM Dichloromethane

DIEA Diisopropylethylamine

DMAP 4-Dimethylaminopyridine

DMF N,N-Dimethylformamide

EtOAc Ethyl acetate

Et₃N Triethylamine

EtOH Ethanol

Et₂O Diethyl ether

LC Liquid chromatography

HOAc Acetic acid

HPLC High performance liquid chromatography

MeCN Acetonitrile

MeOH Methanol

NT 3-Nitro-1,2,4-triazole

on Over night

Pg Protecting group

Ph Phenyl

rt Room temperature

TEST bis(triethoxysilyl)propyl-tetrasulfide

THF Tetrahydrofuran

TFA Trifluoroacetic acid

TFAA Trifluoroacetic anhydride

TIPS Triisopropylsilyl

Preparation of Troxacitabine

Step 1) ((2,2-dimethoxyethoxy)methyl)benzene (Tr-1)

To a stirred solution of 2,2-dimethoxyethanol (50 g, 0.471 mol) in DMF(200 mL), benzyl bromide (56.03 mL, 0.471 mol) and NaOH (20.7 g, 0.518mol) were added at 0° C. and the reaction mixture was stirred at roomtemperature for 16 h. After completion of the reaction (TLC), saturatedsodium chloride solution (500 mL) was added and the reaction mixture wasextracted with DCM (1 L), the organic phase was dried (Na₂SO₄) andconcentrated and the afforded crude was purified by silica gel columnchromatography on 60-120 silica as 4-6% EtOAc in hexane to afford thetitle compound (60 g, 60%) as a liquid.

Step 2)(5S)-5-((4S)-2-((benzyloxy)methyl)-1,3-dioxolan-4-yl)-3,4-dihydroxyfuran-2(5H)-one(Tr-2)

L-Ascorbic acid (44.9 g, 0.255 mol) was added to a solution of compoundTr-1 (60 g, 0.306 mol) in dry acetonitrile (898 mL) followed by additionof pTSA monohydrate (15.5 g, 0.076 mol) and the reaction mixture washeated at 90° C. for 1 h. After completion of the reaction (TLC), halfthe volume of the acetonitrile was distilled off and the process wasrepeated twice. Solvent was removed completely and the title compound asa mixture of stereoisomers was obtained (91 g). The product was directlytaken to the next step without further purification.

Step 3)(2R)-2-((4S)-2-((benzyloxy)methyl)-1,3-dioxolan-4-yl)-2-hydroxyaceticacid (Tr-3)

Compound Tr-2 (91.7 g, 0.297 mol) was added to a stirred solution ofK₂CO₃ (86.3 g, 0.625 mol) in H₂O (509 mL) at room temperature. H₂O₂ (80mL, 0.71 mol, 30% v/v) was slowly added and the solution was cooled to0° C. and then stirred for 24 h. The solvent was removed under reducedpressure, EtOH (100 mL) was added and the mixture was heated at refluxfor 30 min, then filtered. EtOH (100 mL) was added to the afforded solidresidue and the mixture was heated at reflux for 30 min (twice). Thecollected filtrates was concentrated under vacuum which gave the titlecompound (90 g) as a solid.

Step 4) (2S,4S)-2-((benzyloxy)methyl)-1,3-dioxolane-4-carboxylic acid(Tr-4a) & (2R,4S)-2-((benzyloxy)methyl)-1,3-dioxolane-4-carboxylic acid(Tr-4b)

Sodium hypochlorite (650 ml, 0.881 mol, 9-10% in water) was added dropwise over a period of 30 min to a vigorously stirred solution ofcompound Tr-3 (90 g, 0.294 mol) and RuCl₃,xH₂O (1.22 g, 0.0058 mol) inwater (ml pH=8 room temperature). The pH was maintained at 8 by additionof 1M NaOH solution. The reaction mixture was stirred for 3 h in roomtemperature then heated at 35° C. for 12 h. After completion of thereaction (TLC), 1.5 N HCl was added to the reaction mixture at 0° C.until pH 6 was reached, then EtOAc (1 L) was added. The organic phasewashed with brine (2×100 mL), dried (Na₂SO₄), filtered and concentrated.The afforded crude was purified by silica gel column chromatography on230-400 silica as 20% EtOAc in P.ether which gave compounds 4a+4b as amixture of isomers. The isomers were then separated by columnchromatography on silica 230-400 using 0.9% MeOH in DCM and 0.1% AcOH asan eluent, which gave the 2R isomer (20 g, 28%)

Step 5) (2S)-2-((benzyloxy)methyl)-1,3-dioxolan-4-yl acetate (Tr-5)

To a solution of compound Tr-4a (33 g, 0138 mol) in acetonitrile (660mL) was added pyridine (13.2 ml) and lead acetate (79.8 g, 0.180 mol)and the mixture was stirred at room temperature for 16 h. Aftercompletion of the reaction (TLC) the reaction mixture was filtered, thefiltrate was concentrated and the residue was taken in EtOAc (500 mL),washed with water (100 mL) and sat. sodium chloride solution (100 mL)and dried over Na₂SO₄. After removal of the solvent the crude waspurified by column chromatography on 60-120 silica as 12-15%EtOAc/Pet.ether gradient which gave the title compound (16 g, 47%) as aliquid.

Step 6) (2S)-2-(hydroxymethyl)-1,3-dioxolan-4-yl acetate (Tr-6)

To a stirred solution of compound Tr-5 (16 g,) in dry methanol (160 mL),Pd/C (3.2 g, 20% w/w) was added the reaction mixture was hydrogenatedfor 3 h. After completion of the reaction (TLC), the reaction mixturewas filtered through celite. The filtrate was concentrated under reducedpressure and the afforded crude title compound (10 g, 97%) was takendirectly to the next step.

Step 7) ((2S)-4-acetoxy-1,3-dioxolan-2-yl)methyl acetate (Tr-7)

To a stirred solution of compound Tr-6 (5.74 g, 0.0354 mol) in pyridine(107 ml), acetic anhydride (8.22 ml, 0.080 mol) was added at 0° C. andthe reaction mixture was stirred at room temperature for 16 h. Aftercompletion of the reaction (TLC), the reaction mixture was quenched withdil.HCl (10 mL) and extracted to EtOAc (100 mL). The organic phase wasseparated, dried (Na₂SO₄), filtered and concentrated. The afforded crudewas purified by column chromatography on 230-400 silica eluted with agradient of 10-15% EtOAc/Pet.ether which gave the title compound (4.97g, 68%) as a liquid.

Step 8)((2S,4S)-4-(4-(benzylamino)-2-oxopyrimidin-1(2H)-yl)-1,3-dioxolan-2-yl)methylacetate (Tr-8a)

A mixture of N-benzoylcytosine (12.1 g, 56.3 mmol), ammonium sulfate(catalytic amount) and hexamethyldisilazane (HMDS) (67.4 ml, 418 mmol)were refluxed for 1 h. The HMDS was removed under reduced pressure at40° C. and the residue was taken in dry 1,2-dichloroethane (57 ml) andadded the solution of compound Tr-7 (5.7 g, 27.9 mmol) in dry1,2-dichloroethane (57 ml) followed by drop-wise addition of TMSOTf(10.2 ml, 45.7 mmol). The reaction mixture was stirred at roomtemperature for 1 h, then aqueous NaHCO₃ solution was added and themixture was stirred for 30 min. The resulting solid was filtered throughcelite and the filtrate was taken in EtOAc (200 mL), washed with water(50 mL) and dried (Na₂SO₄). After removal of the solvent under reducedpressure the crude was purified by column chromatography on 230-400silica using a gradient of 10-15% EtOAc/Pet.ether to afford a mixture ofanomers which was further separated by SFC purification to afford thetitle compound (3 g, 30%) as a white solid.

Step 9)4-amino-1-((2S,4S)-2-(hydroxymethyl)-1,3-dioxolan-4-yl)pyrimidin-2(1H)-one(Tr-9)

A mixture of compound Tr-8a (3 g), saturated methanolic ammonia solution(180 ml) was stirred at room temperature in a sealed tube for 16 h.After completion of the reaction (TLC), solvent was removed underreduced pressure and the crude was purified by column chromatography on230-400 silica eluted with a gradient of 10-13% MeOH in DCM, which gavethe title compound (1.5 g, 85%) as a solid.

¹H NMR 400 MHz DMSO-d₆ δ: 3.63-3.65 (2H), 4.04-4.07 (2H), 4.92-4.94(1H), 5.18-5.21 (1H), 5.72-5.74 (1H), 6.16-6.18 (1H), 7.14 (1H), 7.26(1H), 7.80-7.82 (1H).

Preparation of 5-F-troxacitabine

Step 1)((2S,4R)-4-(4-benzamido-5-fluoro-2-oxopyrimidin-1(2H)-yl)-1,3-dioxolan-2-yl)methylbenzoate (5-F-Tr-1a)&((2S,4S)-4-(4-benzamido-5-fluoro-2-oxopyrimidin-1(2H)-yl)-1,3-dioxolan-2-yl)methylbenzoate (5-F-Tr-1b)

A mixture of 5-fluoro benzoyl cytosine (9.1 g, 39.5 mmol), ammoniumsulfate (catalytic amount) and hexamethyldisilazane (140 ml) wasrefluxed for 14 h. The HMDS was removed under reduced pressure at 40° C.and the residue was taken in dry 1,2-dichloroethane (50 ml) and addedthe solution of compound ((2S)-4-acetoxy-1,3-dioxolan-2-yl)methylbenzoate (7 g, 26.30 mmol) in dry 1,2-dichloroethane (50 ml) followed bythe drop-wise addition of TMS-OTf (11.6 g, 52.6 mmol). The reactionmixture was stirred at room temperature for 2 h, then aqueous NaHCO₃solution was added to the reaction mixture and the mixture was stirredfor an additional 30 min. The resulting solid was filtered throughcelite and the filtrate was taken in EtOAc (500 mL), washed with water(50 mL) and dried (Na₂SO₄). The solvent was removed under reducedpressure and the crude was purified by column chromatography on 230-400silica as 50-60% EtOAc/Pet.ether gradient to afford pure title compound(1.7 g, 18%) as a solid.

Step 2)4-amino-5-fluoro-1-((2S,4S)-2-(hydroxymethyl)-1,3-dioxolan-4-yl)pyrimidin-2(1H)-one(5-F-Tr)

A mixture of compound 5-F-Tr-1b (1.7 g), saturated methanolic ammoniasolution (34 ml) was stirred at room temperature in a sealed tube for 16h, then the solvent was removed under reduced pressure and the crude waspurified by column chromatography on 230-400 silica as 5% MeOH in DCMgradient to afford the title compound (0.8 g, 68%) as a solid.

The following phenols were prepared and used in the preparation ofintermediates to the compounds of the invention:

Phenol 1

Step a) 1-(3-((Tert-butyldimethylsilyl)oxy)phenyl)ethanone (Ph1-a)

Imidazole (4.46 g, 65.5 mmol) was added to a solution of3-hydroxyacetophenone (4.46 g, 32.8 mmol) in DMF (6 mL). After 5 min, asolution of TBDMS-Cl (4.69 g, 31.1 mmol) in DMF (4 mL) was added. Thereaction mixture was stirred at room temperature for 90 min, then pouredinto hexane containing 5% EtOAc (200 mL) and washed with 1M HCl (60 mL),water (60 mL), saturated sodium bicarbonate (2×60 mL), water (60 mL) andbrine (60 mL). The organic layer was dried over Na₂SO₄, filtered andconcentrated and the afforded residue was purified by flashchromatography on silica gel eluted with hexane/EtOAc, which gave thetitle compound (5.7 g, 69%).

Step b) Tert-butyldimethyl(3-(prop-1-en-2-yl)phenoxy)silane (Ph1-b)

Methyl(triphenylphosphonium)bromide (10.2 g, 28.4 mmol) was suspended indry THF (30 mL) under nitrogen and the suspension was cooled to 0° C.n-Butyllithium (17.8 mL, 28.4 mmol) was added drop-wise to the mixtureand the resulting solution was stirred at room temperature for 30 min.Ph1-a (5.7 g, 22.8 mmol) was added to the mixture and the reactionallowed to proceed at room temperature for 60 min. The reaction wasquenched with aqueous sodium bicarbonate and extracted with diethylether (50 mL). The organic layer was washed with sodium bicarbonatesolution, dried (Na₂SO₄), filtered and concentrated. The affordedresidue was purified through a plug of silica-gel using eluted withhexane, which gave the title compound (3.9 g, 69%).

Step c) tert-butyldimethyl(3-(1-methylcyclopropyl)phenoxy)silane (Ph1-c)

Diethylzinc in hexane (439.2 mmol) was added drop-wise under nitrogenduring 10 minutes to a cooled (0° C.) solution of the olefin Ph1-b (3.9g, 15.7 mmol) in 1,2-dichloroethane (60 mL). Diiodomethane (6.32 mL,78.5 mmol) was added drop-wise and the resulting mixture was stirred at0° C. for 30 min and then allowed to attain room temperature overnight.The mixture was poured into an ice-cold solution of ammonium chlorideand extracted with diethyl ether. The organic layer was washed withsaturated sodium bicarbonate, dried (Na₂SO₄), filtered and concentrated.The crude was taken into hexane and the remaining diiodomethane wasdiscarded. The hexane layer was concentrated to a crude that was takeninto the next step without further purification.

Step d) 3-(1-Methylcyclopropyl)phenol (Phenol 1)

Ph1-c (3.45 g, 13.1 mmol) was taken into 1M solution oftetrabutylammonium fluoride in THF (20 mL, 20 mmol) and the resultingsolution was stirred at room temperature overnight. The reaction wasquenched with 1M HCl (50 mL) and extracted with ethyl acetate (100 mL).The organic layer was washed with brine (2×50 mL), dried (Na₂SO₄),filtered and concentrated. The residue was purified by flashchromatography on silica gel eluted with a mixture of 2-propanol, EtOAcand hexane, which gave the title compound (0.56 g, 29%). MS 147.1[M−H]⁻.

Phenol 2

The title compound was prepared from 4-hydroxyacetophenone (6.0 g, 44.1mmol) using the method described for the preparation of Phenol 1. Yield53%.

Phenol 3

Step a) 1-(3-(benzyloxy)phenyl)cyclopentanol (Ph3-a)

Iodine, warmed up with magnesium, was added to a suspension of magnesiumtunings (1.29 g, 52.8 mmol) in dry THF (50 mL). The mixture was refluxedand about 5% of a solution of 3-bromophenol (13.9 g, 52.8 mmol) wasadded. When the reaction had started, the solution of the bromide wasadded drop-wise and the mixture was then refluxed for one more hour. Themixture was cooled down to about 5° C. and a solution of thecyclopentanone (4.44 g, 52.8 mmol) in THF (50 mL) was added drop-wise.The mixture was stirred at rt for 72 h, then the reactio was quenchedwith cooled saturated ammonium chloride solution and extracted withdiethyl ether (×3). The organic phase was washed with brine, dried(Na₂SO₄), filtered and concentrated. The product was purified by silicagel chromatography (isohexane/EtOAc), which gave the title compound (8.5g, 54%).

Step b) 1-(benzyloxy)-3-(cyclopent-1-en-1-yl)benzene (Ph3-b)

p-Toluenesulfonic acid was added to a solution of Ph3-a (8.4 g, 28.2mmol) in benzene (100 mL). The mixture was refluxed for three hours witha DMF trap, then cooled to rt, diluted with diethyl ether and washedwith a saturated solution of sodium hydrogen carbonate and brine. Theorganic phase was dried (Na₂SO₄), filtered and concentrated. The productwas purified by silica gel chromatography (isohexane/EtOAc), which gavethe title compound (6.45 g, 91%). MS 249.4 [M−H]⁻.

Step c) 3-Cyclopentylphenol (Phenol 3)

A solution of Ph3-b (6.4 g, 26 mmol) in EtOAc (75 mL) and EtOH (75 mL)was hydrogenated at 22° C. and 40 PSI in the presence of 10% Pd oncarbon (1.5 g) in a Parr overnight. The catalyst was filtered off andwashed with EtOAc and EtOH. The solvent was evaporated under reducedpressure and the product was isolated by silica gel chromatography(isohexane/EtOAc), which gave the title compound (3.6 g, 82%). MS 161.2[M−H]⁻.

Phenol 4

Step a) Tert-butyl(3-cyclopropylphenoxy)dimethylsilane (Ph4-a)

A suspension of (3-bromophenoxy)(tert-butyl)dimethylsilane (5.46 g, 19mmol), cyclopropylboronic acid (2.12 g, 24.7 mmol), potassium phosphate,tribasic (14.1 g, 66.5 mmol), tricyclohexylphosphine (0.53 g, 1.9 mmol)and Pd(OAc)₂ (0.21 g, 0.95 mmol) in toluene (80 mL) and water (4 mL) wasstirred at 110° C. overnight. The slurry was diluted with diethyl etherand washed with water and brine. The organic phase was dried (MgSO₄),filtered and concentrated. The crude was purified by flash columnchromatography (EtOAc/hexane) which gave the title compound (1.94 g,41%).

Step b) 3-Cyclopropylphenol (Phenol 4)

1M tetrabutylammonium fluoride (10.1 ml, 10.1 mmol) was added to asolution of Ph4-a (1.94 g, 7.81 mmol) in THF (25 ml). The solution wasstirred for 2 hours, then the solvent was evaporated and the residuedissolved in EtOAc and washed twice with concentrated NH₄Cl (aq) andonce with brine. The organic phase was dried (MgSO4), filtered andconcentrated. The crude was purified by flash column chromatography(hexane/ethyl acetate 9:1 with 1% isopropanol) which gave slightlyimpure title compound (1.24 g, 119%).

Phenol 5

Step a) 2-(4-Bromophenoxy)tetrahydro-2H-pyran(Ph5-a)

4-Bromphenol (3.75 g, 21.7 mmol) was dissolved in 3,4-dihydro-2H-pyran(16 ml, 175 mmol), a catalytic amount of p-Toluenesulfonic acid (15 mg,0.09 mmol) was added and the mixture was stirred at 22° C. for 45 min.The mixture was diluted with diethyl ether and washed with 1 M NaOH (aq)×2, water, dried (Na₂SO₄) and concentrated which gave the title compound(5.57 g, 99%).

Step b) 2-(4-Cyclopropylphenoxy)tetrahydro-2H-pyran (Ph5-b)

A solution of 0.5 M cyclopropyl magnesium bromide in THF (6.5 ml, 3.25mmol) was added during 15 min to a solution of Ph5-a (552.5 mg, 2.15mmol), ZnBr (144 mg, 0.64 mmol), tri-tert-butylphosphinetetrafluoroborate (35.6 mg, 0.12 mmol) and Pd(OAc)₂ (29.5 mg, 0.13 mmol)in THF (4 ml). The mixture was stirred at 22° C. for 90 min then cooledon an ice bath and ice water (10 ml) was added. The mixture wasextracted with EtOAc ×3 and the extracts washed with brine and thendried (Na₂SO₄), filtered and concentrated. The residue was purified bycolumn chromatography on silica (petroleum ether/EtOAc) which gave thetitle compound (292 mg, 62%).

Step c) 4-Cyclopropylphenol (Phenol 5)

p-Toluenesulfonic acid monohydrate (18.9 mg, 0.1 mmol) was added to asolution of Ph5-b (2.28 g, 10.45 mmol) in MeOH (15 ml). The mixture washeated at 120° C. for 5 min in a microwave reactor, then concentratedand purified by column chromatography on silica (petroleum ether/EtOAc).The afforded solids were crystallized from petroleum ether which gavethe title compound (1.08 g, 77%).

Phenol 6

Step a) 1-(3-Methoxyphenyl)cyclobutanol (Ph6-a)

A 1 M solution of 3-methoxyphenyl magnesium bromide in THF (2.11 g, 99.8mmol) was added dropwise between 0 and 10° C. to a stirred solution ofcyclobutanone (6.66 g, 95 mmol) in diethyl ether (65 mL). The mixturewas stirred for three hours at 0-10° C., then the mixture was added toan ice cooled solution of saturated NH₄Cl (300 mL) and water (300 mL).The mixture was stirred for 10 min then extracted three times withdiethyl ether. The organic phase was dried, (Na₂SO₄), filtered andconcentrate. The afforded crude product was purified by silica gelchromatography (isohexane/EtOAc), which gave the title compound (16.9 g,86%).

Step b) 1-cyclobutyl-3-methoxybenzene (Ph6-b)

10% Pd on carbon (2.5 g) was added to a solution of Ph6-a (15.4 g, 86.1mmol) in ethanol (200 mL) and the mixture was hydrogenated in a Parr at60 psi. After 18 h, additional 10% Pd on carbon (1.5 g) was added andthe mixture was hydrogenated for further 18 hours at 60 psi. Thecatalyst was filtered of and washed with EtOH and EtOAc. The solutionwas concentrated under reduced pressure and the crude product wasisolated by silica gel chromatography (isohexane/EtOAc), which gave thetitle compound (14.0 g, 77%).

Step c) 3-cyclobutylphenol (Phenol 6)

A solution of 1M boron tribromide (18.1 g, 72.2 mmol) in DCM was addeddropwise at 0° C. to a solution of Ph6-b (10.6 g, 65.6 mmol) in dry DCM(65 mL). The mixture was stirred for 2.5 hours at −5° C., then thereaction was quenched with cooled saturated solution of NH₄Cl andextracted three times with DCM. The organic phase was dried (Na₂SO₄),filtered and concentrate. The afforded crude product was purified bysilica gel chromatography (isohexane/EtOAc), which gave the titlecompound (9.73 g, 88%).

Phenol 7

Step a) 1-(4-(benzyloxy)phenyl)cyclobutanol (Ph7-a)

A solution of 1-(benzyloxy)-4-bromobenzene (2.63 g, 100 mmol) in diethylether:THF 1:1 (100 mL) was added dropwise at reflux during ≈1 h to asuspension of magnesium tunings (2.43 g) and a trace iodine in diethylether (50 mL). When the addition was completed, the mixture was refluxedfor four hours, then cooled to ≈0° C. Dry THF (50 ml) was added followedby slow addition of a solution of cyclobutanone (7.01 g, 100 mmol) indiethyl ether (50 mL) and the mixture was left to attain rt. Afterstirring for two h, a cool saturated solution of NH₄Cl (500 ml) wasadded and the mixture was stirred for 15 minutes, then extracted twicewith EtOAc. The organic phase was washed with brine, dried with sodiumsulfate and evaporated under reduced pressure. The product was purifiedby column chromatography on silica gel, which gave the title compound(12.5 g, 42%).

Step b) 4-cyclobutylphenol (Phenol 7)

Pd 10% on carbon (2.55 g, 21.5 mmol) was added under argon to a solutionof Ph7-a (12.4 g, 41.4 mmol) in abs EtOH (110 mL) the and the mixturewas hydrogenated at 45 psi at rt for 18 h. The catalyst was filtered of,washed with ethanol and the solution was concentrated. The product waspurified by silica gel chromatography (isohexane—EtOAc). Appropriatefractions were pooled and concentrated and the residue crystalized frompetrol ether which gave the title compound (3.15 g, 51%).

Phenol 8

4-(1-Methylcyclopentyl)phenol (Phenol 8)

A solution of 1-methylcyclopentanol (2.00 g, 20.0 mmol) and phenol (2.07g, 22.0 mmol) in pentane (50 mL) were added dropwise during 30 min to asuspension of fresh AlCl₃ (1.33 g, 10 mmol) in pentane (100 mL). Theresulting mixture was stirred under N₂ at rt for 72 h, then the reactionmixture was poured into water/ice and HCl (12 M, 20 mmol, 1.66 mL). Theorganic phase was washed with water (50 mL) and brine (50 mL), dried(Na₂SO₄) filtered and concentrated. The crude was purified by columnchromatography on silica (MeOH—DCM), which gave the title compound (426mg, 12%).

Phenol 9

Step a) 2-(4-Bromo-3-methylphenoxy)tetrahydro-2H-pyran (Ph9-a)

pTs (16 mg, 0.086 mmol) was added to a solution of4-bromo-3-methylphenol (4.0 g, 21.4 mmol) in 3,4-dihydro-2-H-pyran (16mL, 175 mmol). The reaction mixture was stirred at room temperature for1 h, then diluted with diethyl ether and washed with 1M NaOH (aq) andwater. The organic phase was dried (Na₂SO₄) filtered and concentrated.The crude was purified by column chromatography on silica(EtOAc/heptane) which gave the title compound (3.32 g, 57%).

Step b) 2-(4-Cyclopropyl-3-methylphenoxy)tetrahydro-2H-pyran (Ph9-b)

Ph9-a (3.12 g, 11.5 mmol), ZnBr₂ (2.59 g, 11.5 mmol),tri-tert-butylphosphine tetrafluoroborate (0.2 g, 0.69 mmol) andPd(OAc)₂ (258 mg, 1.15 mmol) were put in a flask and the flask wasflushed with N₂ a couple of times. THF (10 mL) was added while stirring,followed by dropwise addition of 0.5 M cyclopropylmagnesium bromide inTHF (35 mL, 17.4 mmol) during 5 minutes. The mixture was stirred at rton, then filtered through a Celite plug, eluted with MeOH. The solutionwas concentrates and the crude was purified by column chromatography onsilica (EtOAc/heptane) which gave the title compound (1.69 g, 57%).

Step c) 4-Cyclopropyl-3-methylphenol (Phenol 9)

Ph9-b (1.70 g, 7.30 mmol) was dissolved in MeOH (20 ml) and pTsxH₂O (318mg, 1.67 mmol) was added. The mixture was stirred at 22° C. for 30minutes, then concentrated. The crude was purified by columnchromatography (EtOAc/heptane), which gave the title compound (704 mg,65%).

Phenol 10

Step a) 4-cyclopropyl-1-methoxy-2-methylbenzene (Ph10-a)

4-Bromo-1-methoxy-2-methylbenzene (4.39 g, 21.9 mmol) was reacted withcyclopropylmagnesium bromide according to the procedure described in Ph9step b, which gave the title compound (1.54 g, 43%).

Step b) 4-cyclopropyl-2-methylphenol (Phenol 10)

BBr₃ (5 mL, 5 mmol) was added under N₂ at 0° C. to a solution of Ph10-a(1.54 g, 9.49 mmol) in DCM (7.5 mL). The reaction was stirred for 2 h,then quenched with MeOH (3 mL) and concentrated. The crude was dissolvedin EtOAc and washed with brine. The organic phase was dried (Na₂SO₄),filtered and concentrated. The crude product was purified by columnchromatography on silica, which gave the title compound (826 mg, 59%).MS 147.11 [M−H]⁻.

Phenol 11

4-cyclopropyl-3-methoxyphenol (Phenol 11)

The title compound was prepared from 4-bromo-3-metoxyphenol (1.11 g,5.49 mmol) according to the procedure described for the preparation ofPhenol 9. Yield 40%.

Phenol 12

Step a) 3-(dimethylamino)-1-(3-hydroxyphenyl)propan-1-one (Ph12-a)

A few drops of HCl were added to a solution of 3-hydroxy acetophenone(4.08 g, 30 mmol), paraformaldehyde (4.05 g, 45 mmol) and dimethylaminehydrochloride (2.69 g, 33 mmol) in absolute EtOH (100 mL) and thereaction mixture refluxed for 18 h. Additional dimethylaminehydrochloride (0.55 eq., 1.22 g), paraformaldehyde (0.5 eq., 1.35 g) andHCl (0.5 mL) were added and the reaction mixture refluxed for additional4 h, then cooled to rt. The precipitated white solid was collected andwashed with cold EtOH (50 mL) and cold acetone (10 mL) and then freezedried, which gave the title compound (2.59 g, 38%) that was used in thenext step without further purification.

Step b) cyclopropyl(3-hydroxyphenyl)methanone (Phenol 12)

NaH (60% mineral oil dispersion) (1.13 g, 28.2 mmol) was added inportions at rt to a stirred suspension of trimethylsulfoxonium iodide(6.20 g, 28.2 mmol) in DMSO (100 mL). After 1 h, solid Ph12-a (2.59 g,11.3 mmol) was added in portions under stirring and cooling. Thereaction mixture was stirred at rt for 40 h, then poured into cold water(200 mL) and extracted with DCM (3×100 mL). The organic phase was washedwith a saturated aqueous solution of NH₄Cl (2×100 mL), dried (Na₂SO₄),filtered and concentrated. The afforded crude was purified by columnchromatography on silica (MeOH/DCM) which gave the title compound (883mg, 48%).

Phenol 13

Step a) cyclopropyl(4-hydroxyphenyl)methanone (Ph13)

p-Hydroxy-γ-chlorobutyrophenone (4.95 g) was added in portions duringapproximately 30 min to a solution of NaOH (8 mL, aq, 50% w/w), thenNaOH (35 mL, aq, 25% w/w) was added followed by p-hydroxyγ-chlorobutyrophenone (4.95 g) in one portion. The temperature waslowered to 140° C. and NaOH (8 g) was added. After 90 min, H₂O (10 mL)was added, and after additional 60 min, the reaction mixture was cooled,diluted with H₂O and neutralized with HOAc (≈27-30 ml) to pH≈7 Theformed precipitate was filtered, washed with H₂O and dried in vacuum.The solids were triturated in CHCl₃ (200 ml) at 40° C. during 10 min,then at RT overnight. The slurry was heated to 40° C. during 30 min,then filtered. The filtrate was dried (MgSO₄), filtered and concentratedto ≈70 ml. Hexane was added and an oil was formed that eventually becamecrystals. The slurry was filtered, solids washed with CHCl₃/hexane anddried, which gave the title compound (4.15 g, 51%).

Phenol 14

Step a) 3-(1-hydroxy-2,2-dimethylpropyl)phenol (Ph14-a)

t.Bu-MgBr (1.5 eq.) was added dropwise during 30 minutes to a cold (−10°C.) mixture of 3-hydroxybenzaldehyde (2.00 g, 16.4 mmol) in diethylether (20 mL). During the addition THF (20 mL) was added. The mixturewas allowed to reach 23° C. and stirred for 6 hours. More t.Bu-MgBr (0.7eq.) was added and the mixture was left stirring over night, then cooledand the reaction was quenched with aqueous saturated NH₄Cl. to give.EtOAc was added to the mixture followed by addition of 1 M aqueous HCluntil a homogeneous mixture was obtained. The phases were separated andthe organic phase was washed with brine, dried (Na₂SO₄), filtered andconcentrated. The afforded crude was purified by column chromatography,which gave the title compound (1.1 g, 37%).

Step b) 1-(3-hydroxyphenyl)-2,2-dimethylpropan-1-one (Ph14)

To an oven dried round bottomed flask was added 3 Å MS and pyridiniumchlorochromate (PCC) (1.97 g, 9.15 mmol) followed by dry DCM (5 mL). Themixture was stirred at 20° C. for 5 minutes whereafter a mixture ofAA8019 (1.10 g, 6.10 mmol) in DCM (5 mL) was added slowly. Aftercomplete oxidation the mixture was filtered through a pad of Celite,washing the pad with diethyl ether. The filtrate was concentrated. Thecrude was purified by column chromatography which gave the titlecompound (402 mg, 37%). MS 179.25 [M+H]+.

Phenol 15

1-(4-Hydroxyphenyl)-2,2-dimethylpropan-1-one (Ph15)

4-hydroxybenzaldehyde (3 g, 24.6 mmol) was reacted according to theprocedure described for the preparation of Phenol 14, which gave thetitle compound (538 mg, 17%).

Amino Acid 1

Step a) (S)—(S)-sec-butyl 2-((tert-butoxycarbonyl)amino)propanoate(AA1-a)

L-Boc-Alanine (2.18 g, 11.5 mmol) was dissolved in dry DCM (40 mL) andthe alcohol (R)-butan-2-ol (938 mg, 12.6 mmol) was added. The mixturewas cooled to about 5° C. and EDC (3.31 g, 17.2 mmol) was added in oneportion followed by portionwise addition of DMAP (140 mg, 1.15 mmol).The mixture was allowed to attain room temperature and stirredovernight, then diluted with ethyl acetate (˜300 ml) and the organicphase was washed three times with a saturated solution of sodiumhydrogen carbonate and once with brine. The organic phase was dried oversodium sulfate and concentrated under reduced pressure. The product wasisolated by silica gel chromatography eluted with isohexane and 10%ethyl acetate, which gave the title compound (2.78 g, 98%).

Step b) (S)—(S)-Sec-butyl 2-aminopropanoate (AA1-b)

A mixture of AA1-a (2.77 g, 11.3 mmol) and p-toluene sulfonic acid monohydrate (2.15 g, 11.3 mmol) in EtOAc (45 mL) was stirred for 16 h at 65°C., then concentrated under reduced pressure. The afforded residue wascrystallised from diethyl ether, which gave the title compound (3.20 g,89%).

Amino Acid 2

(S)—(R)-Pentan-2-yl 2-aminopropanoate (AA2)

The procedure described for the preparation of AA1 was followed butusing (R)-pentan-2-ol instead of (R)-butan-2-ol, which gave the titlecompound (4.6 g).

Amino Acid 3

(S)—(S)-Pentan-2-yl 2-aminopropanoate (AA3)

The procedure described for the preparation of AA1 was followed butusing (S)-pentan-2-ol instead of (R)-butan-2-ol, which gave the titlecompound (8.3 g).

The following intermediates were prepared and can be used in thepreparation of compounds of the invention:

Intermediate 1

Step a) (R)-4-fluorobenzyl 2-((tert-butoxycarbonyl)amino)propanoate(I-1a)

Boc-L-AlaOH (19.92 mmol), DMAP(1.99 mmol) and (4-fluorophenyl)methanol(23.9 mmol) were dissolved in CH₂Cl₂ (100 mL). To this solution wasadded triethylamine (23.9 mmol) followed by EDCl (23.9 mmol) and theresulting reaction mixture was stirred overnight at room temperatureunder N₂. The reaction mixture was diluted with CH₂Cl₂ (100 mL), washedwith saturated aqueous solution of NaHCO₃ (2×50 mL), saturated aqueoussolution of NaCl (2×50 mL), dried (Na₂SO₄) and concentrated. Theafforded residue was purified by column chromatography on silica geleluted with n-hexane-EtOAc (95:5 to 60:40) which gave the title compound(4.44 g) as a white waxy solid. MS: 296 [M−H]⁻.

Step b) (R)-4-fluorobenzyl 2-aminopropanoate (I-1b)

Compound I-1a (14.93 mmol) was dissolved in 4M HCl/dioxane (40 mL) andstirred at room temperature for 30 minutes and evaporated to drynesswhich gave the hydrochloride salt of the title compound (3.4 g) as awhite powder. MS: 198 [M+H]⁺.

Step c) (2R)-4-fluorobenzyl2-((chloro(phenoxy)phosphoryl)amino)propanoate (I-1)

PhOPOCl₂ (4.28 mmol) was added dropwise at −78° C. to a solution ofcompound I-5b (4.28 mmol) in CH₂Cl₂ followed by dropwise addition oftriethylamine (8.56 mmol). The resulting reaction mixture was stirred at−78° C. under Ar and allowed to attain room temperature overnight. Thereaction mixture was evaporated on silica gel and purified bychromatography (n-hexane/EtOAc (88:12)-(0:100)). which gave the titlecompound (769 mg). ³¹P-NMR (CDCl₃) δ: 7.85 (s) and 7.54 (s) (R_(P) andS_(P) diastereomers).

Intermediate 2

Step a) (S)—(R)-sec-butyl 2-((tert-butoxycarbonyl)amino)propanoate(I-2a)

L-Boc-Alanine (2.18 g, 11.5 mmol) was dissolved in dry DCM (40 mL) andthe alcohol (R)-butan-2-ol (938 mg, 12.6 mmol) was added. The mixturewas cooled to about 5° C. and EDC (3.31 g, 17.2 mmol) was added in oneportion followed by portionwise addition of DMAP (140 mg, 1.15 mmol).The mixture was allowed to attain room temperature and stirredovernight, then diluted with ethyl acetate (˜300 ml) and the organicphase was washed three times with a saturated solution of sodiumhydrogen carbonate and once with brine. The organic phase was dried oversodium sulfate and concentrated under reduced pressure. The product wasisolated by silica gel chromatography eluted with isohexane and 10%ethyl acetate, which gave the title compound (2.78 g, 98%).

Step b) (S)—(R)-Sec-butyl 2-aminopropanoate (I-2b)

A mixture of I-10a (2.77 g, 11.3 mmol) and p-toluene sulfonic acid monohydrate (2.15 g, 11.3 mmol) in EtOAc (45 mL) was stirred for 16 h at 65°C., then concentrated under reduced pressure. The afforded residue wascrystallised from diethyl ether, which gave the title compound (3.20 g,89%).

Step c) (2S)—(R)-Sec-butyl2-(((4-nitrophenoxy)(phenoxy)phosphoryl)amino)propanoate (I-2)

Phenyl dichlorophosphate (1 eq) was added under nitrogen at −30° C. to asolution of Compound I-10b (3.15 g, 9.92 mmol) in DCM (75 ml), followedby dropwise addition of triethylamine (2 eq). The mixture was allowed toattain room temperature and stirred overnight, then cooled to about 5°C. and 4-nitrophenol (1 eq, 15 mmol) was added as a solid followed bydropwise addition of triethylamine (1 eq g, 15 mmol) and the mixture wasstirred for 4 hours at room temperature, then concentrated under reducedpressure, diluted with ethyl acetate (40 ml) and ether (40 ml) and leftat room temperature overnight. The triethylamine-HCl salt was filteredof and the filtrate was concentrated under reduced pressure. Theafforded residue was purified by column chromatography on silica geleluted with iso-hexane-ethyl acetate, which gave the title compound(4.19 g, 79%).

The following compounds were prepared according to the proceduredescribed for the preparation of I-2 using the appropriate alcohol:

I-# Structure alcohol I-3

cyclopropylmethanol I-4

cyclopentylmethanol I-5

pentan-3-ol I-6

2-propylpentan-1-ol

Intermediate 6, Diastereomer-1 & -2

The two diastereomers of compound I-6 were separated by SFC, which gaveI-6-dia-1 and I-6-dia-2.

Intermediate 7

Step a) (S)-cyclooctyl 2-aminopropanoate (I-7a)

To a slurry of L-alanine (1.7 g, 19.1 mmol) and cyclooctanol (25 ml, 191mmol) in toluene (100 ml) was added p-toluenesulfonic acid monohydrate(3.6 g, 19.1 mmol). The reaction mixture was heated at refluxtemperature for 25 h and water was removed from the reaction using aDean-Stark trap. The mixture was concentrated under reduced pressure andthe residue kept under vacuum over night. To the residue (27 g) wasadded diethyl ether (100 ml). The white precipitate was collected byfiltration, washed with diethyl ether (3×50 ml) and dried under vacuumwhich gave the title compound (4.84 g, 68%).

Step b) (2S)-cyclooctyl2-(((4-nitrophenoxy)(phenoxy)phosphoryl)amino)propanoate (I-7)

Compound I-7a was reacted according to the method described for thepreparation of I-2 step c, which gave the title compound (4.7 g, 76%)

Intermediate 8

(2S)-cycloheptyl2-(((4-nitrophenoxy)(phenoxy)phosphoryl)amino)propanoate(I-22)

The procedure described for the preparation of compound I-7 was followedbut using cycloheptanol (27 ml, 224 mmol) instead of cyclooctanol, whichgave the title compound (5.72 g, 55%).

Intermediate 9

(2S)-Cyclohexyl 2-(((4-nitrophenoxy)(phenoxy)phosphoryl)amino)propanoate(I-23)

The procedure described for the preparation of I-2 step c was followedbut using (S)-cyclohexyl 2-aminopropanoate instead of(S)-3,3-dimethylbutyl 2-aminopropanoate, which gave the title compound(10.6 g, 82%).

Intermediate 10

(S)-2-Ethylbutyl 2-((bis(4-nitrophenoxy)phosphoryl)amino)propanoate(I-10)

(S)-2-Ethylbutyl 2-aminopropanoate (5 g, 14.49 mmol) was added to asolution of bis(4-nitrophenyl) phosphorochloridate (6.14 g, 17.1 mmol)in DCM (50 ml), the mixture was cooled in an ice bath and Et₃N (4.77 mL,34.2 mmol) was added drop wise. The cooling was removed after 15 min andthe reaction mixture was stirred at 23° C. until complete reactionaccording to TLC. Diethyl ether was then added, the mixture was filteredand the filtrate was concentrated and purified by column chromatographyon silica which gave the title compound (2.05 g, 82%).

Intermediate 11

Step a) (S)-isopropyl 2-aminopropanoate (I-11a)

SOCl₂ (29 mL, 400 mmol) was added dropwise at 0° C. to a suspension ofthe HCl salt of L-alanine (17.8 g, 200 mmol) in isopropanol (700 mL).The suspension was stirred at room temperature over night, thenconcentrated, which gave the title compound (29.2 g, 87%).

Step b) (2S)-Isopropyl2-(((((S)-1-isopropoxy-1-oxopropan-2-yl)amino)(4-nitrophenoxy)phosphoryl)-amino)propanoate(I-11)

A solution of 4-nitrophenyl dichlorophosphate (1.8 g 7 mmol) in DCM wasadded dropwise at −60° C. to a solution of the amine I-11a (2.35 g, 14mmol) and triethylamine (7.7 mL, 56 mmol) in DCM. The reaction mixturewas allowed to attain room temperature, stirred over night, concentratedand then diluted with ethyl acetate and ether and left at roomtemperature overnight. The triethylamine-HCl salt was filtered of, thefiltrate was concentrated under reduced pressure and the affordedresidue was purified by chromatography on silica gel eluted withiso-hexane-ethyl acetate, which gave the title compound (1.6 g, 50%).

Intermediate 12

Step a) (S)-Neopentyl 2-((tert-butoxycarbonyl)amino)propanoate (I-12a)

EDAC and DMAP was added in portions at −5° C. to a solution ofBoc-alanine (18.9 g, 100 mmol) and neopentylalcohol (13.0 mL, 120 mmol)in DCM (200 mL). The reaction mixture was allowed to attain roomtemperature and stirred for 72 h. EtOAc (700 mL) was added and theorganic phase was washed three times with a saturated solution of NaHCO₃and once with brine, then concentrated. The afforded residue waspurified by column chromatography eluted with hexane-EtOAc 90/10 to80/20, which gave the title compound (21 g, 81%).

Step b) (S)-Neopentyl 2-aminopropanoate (I-12b)

p-Toluene sulfonic acid (15.6 g, 82.0 mmol) was added at −65° C. to asolution of the Boc protected amine I-12a (21.1 g, 82.0 mmol) in EtOAc(330 mL). The reaction mixture was stirred at −65° C. for 8 h, then leftto attain room temperature overnight. The mixture was then filtered andconcentrated which gave the title compound (21 g, 78%).

(2S)-Neopentyl2-(((((S)-1-(neopentyloxy)-1-oxopropan-2-yl)amino)(4-nitrophenoxy)-phosphoryl)amino)propanoate(I-12)

4-Nitrophenol dichlorophosphate was added dropwise during 1 h at −50° C.to a solution of the amine I-12b (3.90 g, 24.5 mmol) in DCM (100 mL).The reaction mixture was allowed to attain room temperature, stirredovernight, concentrated and then diluted with diethyl ether and left atroom temperature overnight. The mixture was filtered, the filtrate wasconcentrated under reduced pressure and the afforded residue waspurified by chromatography on silica gel eluted with iso-hexane-ethylacetate, which gave the title compound (4.8 g, 77%).

Intermediate 32

(2S)—(R)-sec-butyl2-(((perfluorophenoxy)(phenoxy)phosphoryl)amino)propanoate (I-32)

Et₃N (10.9 mL, 78.1 mmol) was added dropwise at −70° C. under nitrogenduring 15 minutes to a stirred solution of the pTs salt of(S)—(R)-sec-butyl 2-aminopropanoate (12.0 g, 37.7 mmol) in DCM (50 mL).To this mixture was added a solution of phenyl dichlorophosphate (5.61mL, 37.7 mmol) in DCM (50 mL) during 1 h. The reaction mixture wasstirred at −70° C. for additional 30 minutes, then allowed to warm to 0°C. during 2 h and stirred for 1 h. A solution of pentafluorophenol (6.94g, 37.7 mmol) and Et₃N (5.73 mL, 41.1 mmol) in DCM (30 mL) was added tothe mixture during 20 minutes. The crude mixture was allowed to stir at0° C. for 18 h, and was then concentrated. The residue was taken in THF(100 mL), insolubles were filtered off and washed several times withTHF. The solvent was evaporated and the residue triturated withtert.butyl methyl ether. Insolubles were filtered off and washed withtert.buty methyl ether. The combined filtrate was concentrated and thecrude solid sonicated with n-hexane/EtOAc (80:20; 100 mL). The solid wasfiltered, washed with n-hexane/EtOAc (80:20) which gave the purephosphorus stereoisomer of the title compound as a white solid (2.3 g,13%).

Intermediate 33

(2S)-ethyl 2-(((perfluorophenoxy)(phenoxy)phosphoryl)amino)propanoate(I-33)

The pure phosphorus stereoisomer of the title compound was preparedaccording to the method described for I-32, but starting from the HClsalt of (S)-ethyl 2-aminopropanoate (11.0 g, 71.1 mmol). Yield 8.56 g,27%.

Intermediate 34

(2S)-2-ethylbutyl2-(((perfluorophenoxy)(phenoxy)phosphoryl)amino)propanoate (I-34)

The pure phosphorus stereoisomer of the title compound was preparedaccording to the method described for 1-32, but starting from the pTssalt of (S)-2-ethylbutyl 2-aminopropanoate (18.8 g, 54.4 mmol). Yield27.0 g, 99%.

LC-MS 496.44 [M+H]⁺.

Intermediate 35

(2S)-butyl 2-(((perfluorophenoxy)(phenoxy)phosphoryl)amino)propanoate(I-35)

Phenyl dichlorophosphate (12.4 mL, 83.1 mmol) was added to a cooled(−20° C.) slurry of (S)-butyl 2-aminopropanoate (26.4 g, 83.1 mmol) indichloromethane (200 mL). The mixture was stirred for 10 min then Et₃N(25.5 mL, 183 mmol) was added dropwise for 15 min. The mixture wasstirred at −20° C. for 1 h then at 0° C. for 30 min. The mixture waskept cooled in an ice-bath and perfluorophenol (15.3 g, 0.08 mol) wasadded followed by a dropwise addition of Et₃N (11.6 mL, 0.08 mol). Themixture was stirred over night and slowly taken to 20° C. Diethyl etherwas added and the mixture was filtered through Celite, concentrated andpurified by column chromatography on silica gel eluted with petroleumether/EtOAc (9:1→8:2). Appropriate fractions were pooled, concentratedand crystallized from petroleum ether EtOAc (9:1) which gave the purephosphorus stereoisomer of the title compound as a white solid (2.23 g,5.8%).

Intermediate 36

Step a) L-Alanine isopropylester hydrochloride (I-36a)

Thionylchloride (80.2 g, 0.674 mol, 1.5 eq) was added with cooling to2-propanol (400 mL) at −7 to 0° C. over a period of 30 minutes, followedby addition of L-alanine (40.0 g, 0.449 mol) at 0° C. A flow indicatorand a scrubber with a mixture of 27.65% sodium hydroxide (228 g) andwater (225 g) were attached to the outlet. The reaction mixture wasstirred at 67° C. for two hours, then at 70° C. for one hour and at20-25° C. over night. The reaction mixture was distilled at 47-50° C.under reduced pressure (250-50 mBar) from a 60° C. bath. When thedistillation became very slow, toluene (100 mL) was added to theresidual oil, and the distillation at 48-51° C. under reduced pressure(150-50 mBar) from a 60° C. bath was continued until it became veryslow. t-butylmethylether (tBME)(400 mL) was added to the residual oil,and the two-phase system ws seeded under efficient stirring at 34-35° C.When crystallization was observed the mixture was cooled to 23° C. overa period of one hour, and the precipitate isolated by filtration. Thefilter cake was washed with tBME (100 mL) and dried to constant weightunder reduced pressure without heating, which gave the title compound(67.7 g, 90%) as white solids.

Step b) (S)-Isopropyl2-(((S)-(perfluorophenoxy)(phenoxy)phosphoryl)amino)propanoate (I-36)

Phenyl dichlorophosphate (62.88 g, 0.298 mol, 1.0 eq) was added undernitrogen to a solution of L-alanine isopropylester hydrochloride (50.0g, 0.298 mol) in DCM (310 mL) at 0° C.—the addition was completed bywash with DCM (39 mL). The mixture was cooled and triethylamine (63.35g, 0.626 mol, 2.1 eq) was added over a period of 70 minutes with coolingkeeping the temperature not higher than −14° C., the addition wascompleted by wash with DCM (39 mL). The mixture was stirred for one hourat −15 to −20° C., then heated to −8° C. and a solution ofpentafluorophenol (60.38 g, 0.328 mol, 1.1 eq) and triethylamine (33.19g, 0.328 mol, 1.1 eq) in DCM (78 mL) was added over a period of 42minutes with cooling keeping the temperature not higher than 0° C.—theaddition was completed by wash with DCM (39 mL). The mixture was stirredfor one hour at 0° C. and then over night at +5° C. The formedprecipitate was removed by filtration, and the filter cake washed withDCM (95 mL). The combined filtrates were washed at 5° C. with water(2×190 mL). The organic phase was distilled at 32-38° C. at reducedpressure (650-600 mBar), and distillation was continued until a residualvolume of approx. 170 mL partly crystallized mass was obtained. Ethylacetate (385 mL) was added, and the resulting clear solution wasdistilled at 43-45° C. under reduced pressure (300-250 mBar).Distillation was continued until a residual volume of approx. 345 mL wasobtained. The clear solution was cooled to 36° C., and crystallizationis induced by addition of seed crystals of (S)-isopropyl2-(((S)-(perfluorophenoxy)(phenoxy)phosphoryl)amino)propanoate (20 mg)prepared as described in J. Org. Chem., 2011, 76, 8311-8319. The mixturewas cooled to 27° C. over a period of one hour, then n-heptane (770 mL)was added over a period of 47 minutes, and the mixture was stirred foran additional period of 37 minutes. Triethylamine (6.03 g, 0.2 eq) wasadded, and the mixture was stirred at 23-25° C. over night. Theprecipitate was isolated by filtration. The filter cake was washed withethyl acetate:n-heptane (1:9, 80 mL) and dried to constant under reducedpressure (below 0.1 mBar) without heating, which gave the title compound(75.64 g, 56%) as a white crystalline material.

¹H NMR (CDCl₃, 300 MHz) δ 7.38-7.32 (m, 2H), 7.27-7.24 (m, 2H),7.23-7.19 (m, 1H), 5.10-4.98 (m, 1H), 4.20-4.08 (m, 1H), 4.03-3.96 (m,1H), 1.46 (dd, 7.2, 0.6 Hz, 3H), 1.26-1.23 (2xd, 6H);

¹³CNMR (CDCl₃, 100 MHz) δ 172.7 (d, J=8.8 Hz), 150.4 (d, J=7.1 Hz),143.4-143.0 (m), 141.0-140.2 (m), 140.0-139.8 (m), 137.6-137.2 (m),136.8-136.2 (m), 130.0 (d, J=0.82 Hz), 125.8 (d, J=1.4 Hz), 120.3 (d,J=5.0 Hz), 69.8, 50.6, (d, J=1.9 Hz), 21.8 (d, J=1.9 Hz), 21.2 (d, J=4.4Hz);

The crystallization properties and NMR spectral data of the titlecompound were in agreement with published data (J. Org. Chem., 2011, 76,8311-8319), thus confirming the S stereochemistry of the phosphorus atomof the title compound.

Intermediate 37

Step a) (S)-Cyclohexyl 2-aminopropanoate (I-37a)

Acetylchloride (4.2 mL, 59.3 mmol) was added drop-wise to a stirredsolution of cyclohexanol (50 ml), followed by L-phenylalanine (4.0 g,24.2 mmol). The reaction mixture was heated to 100° C. for 16 h, thenconcentrated under reduced pressure, triturated with diethylether/Hexane (1:1) and dried to afford the title compound (6 g, 88%) aswhite solid which was used in next step without further purification.

Step b) (S)-Cyclohexyl2-(((S)-(perfluorophenoxy)(phenoxy)phosphoryl)amino)propanoate (I-37)

To a stirred solution of compound I-37a (7.0 g, 24.6 mmol) in dry DCM(42 mL) triethylamine (7.17 mL, 51.5 mmol) was drop wise added at −70°C. over 30 minutes, followed by addition of a solution of phenyldichlorophosphate (5.15 g, 34.5 mmol) in dry DCM (21 mL) over 1 h. Thereaction mixture was stirred at −70° C. for additional 30 min and thenallowed to warm 0° C. over 2 h and stirred for 1 h. To this mixture wasadded a solution of pentafluorophenol (4.94 g, 26.8 mmol) andtriethylamine (3.74 mL, 26.8 mmol) in dry DCM (28 mL) over 1 h. Themixture was allowed to stir at 0° C. for 4 h, and then left at 5° C. for16 h. The reaction mixture was filtered and the filtrate wasconcentrated under reduced pressure. The crude solid was dissolved inEtOAc (300 mL), washed with water (50 mL), dried and the solvent wasremoved under reduced pressure. The obtained solid was triturated with20% EtOAc in hexane, filtered, washed with hexane and dried to affordthe title compound as a single diastereomer (3.0 g, 21%) as a solid.

Intermediate 38

(2S)-Isopropyl 2-(((4-nitrophenoxy)(phenoxy)phosphoryl)amino)propanoate(I-38)

To a stirred solution of 4-nitrophenyldichlorophosphate (5 g, 19.8 mmol)in dry DCM (40 ml) was added a solution of phenol (1.86 g, 19.8 mmol)and triethylamine (3 mL, 21.8 mmol) in dry DCM (50 mL) at −78° C. over aperiod of 30 min. The mixture was stirred at this temperature for 60min, then transferred to another flask containing a solution of compound(S)-isopropyl 2-aminopropanoate (3.3 g, 19.8 mmol) in dry DCM (40 mL) at−5° C. over a period of 15 min. To this mixture was added a secondportion of TEA (6 mL, 43.3 mmol) at −5° C. over a period of 20 min. Themixture was stirred at 0° C. for 3 h, then the solvent was removed underreduced pressure. The residue was taken in EtOAc (200 mL) and washedwith water (50 mL), dried over Na₂SO₄ and the solvents were removedunder reduced pressure to give the crude product as an oil, which waspurified by column chromatography using 0-20% EtOAc/Hexane gradient and230-400 mesh silica gel to give a mixture of diastereomers in about 1:1ratio. The two diastereomers were separated by SFC which gave the titlecompound, Isomer 1 (1.5 g, 20%) and Isomer 2 (1.5 g, 18%) as solids.

The compounds listed in Table 1 were prepared and the diastereomersseparated according to the procedure described for the preparation ofIntermediate I-38, using the appropriate amino acid ester and phenol.

TABLE 1

Example 1

Step a)((2S,4S)-4-(2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)-1,3-dioxolan-2-yl)methylacetate (1a)

A mixture of compound Tr-8 (0.15 g, 0.41 mmol), 1,2-dimethoxyethane (1.5mL) and water (0.96 mL) were heated in the sealed tube at 125° C. for 48h. After completion of the reaction (TLC), the reaction mixture wascooled to room temperature and solvents were removed under reducedpressure. The crude residue was purified by column chromatography on230-400 silica as 3-7% MeOH/DCM gradient which gave compound 1a (0.08 g,80%) as a solid and compound 1b (0.02 g) as a solid.

Step b)1-((2S,4S)-2-(hydroxymethyl)-1,3-dioxolan-4-yl)pyrimidine-2,4(1H,3H)-dione(1b)

Compound 1a (0.08 g, 0.31 mmol) in a saturated solution of NH₃ in MeOH(1.6 mL) was stirred in the sealed tube at room temperature for 4 h.After completion of the reaction (TLC), the solvents were removed underreduced pressure and the residue was purified by column chromatographyon 60-120 silica using 5-7% MeOH/DCM to afford compound the titlecompound (0.06 g, 90%) as a solid.

Step c) (2S)-isopropyl2-(((((2S,4S)-4-(2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)-1,3-dioxolan-2-yl)methoxy)(phenoxy)phosphoryl)amino)propanoate(1c)

To a stirred solution of compound 1b (60 mg, 0.28 mmol) in DMPU (0.6mL), tert-butylmagnesiumchloride (0.57 mL, 0.98 mmol, 1.7 M in THF) wasdrop-wise added at −5° C. The mixture was stirred at −5° C. for 30 min,then at room temperature for 30 min. A solution of isopropyl((perfluorophenoxy)(phenoxy)phosphoryl)-L-alaninate (0.25 g, 0.56 mmol)in dry THF (2.5 mL) was added at −5° C. and the reaction mixture wasstirred at room temperature for 8 h. After completion of the reaction(TLC), water (15 mL) was added and the mixture was extracted with EtOAc(30 mL). The organic phase was washed with sat. sodium chloride solution(10 mL), dried (Na₂SO₄), filtered and concentrated, and the affordedcrude was purified by column chromatography on 230-400 silica as 4-5%MeOH/DCM gradient which gave the title compound (55 mg, 38%) as a solid.MS (ES+) [484.0]⁺.

¹H NMR (DMSO-d₆, 400 MHz) δ 1.15-1.20 (10H), 3.73-3.75 (1H), 4.11-4.27(4H), 4.84-4.90 (1H), 5.14 (1H), 5.51-5.53 (1H), 6.06-6.12 (1H),6.26-6.27 (1H), 7.17-7.23 (3H), 7.36-7.40 (2H), 7.57-7.60 (1H), 11.37(1H).

Example 2

(2S)-Isopropyl2-(((((2S,4S)-4-(4-amino-2-oxopyrimidin-1(2H)-yl)-1,3-dioxolan-2-yl)methoxy)(phenoxy)phosphoryl)amino)propanoate(2)

Troxacitabine (TR-9) (50 mg, 0.23 mmol) was reacted with thephosphorylating agent I-36 (0.26 g, 0.58 mmol) according to theprocedure described in Example 1 step c, which gave the title compound(30 mg, 26%) as a solid. MS (ES+) 483.34 [M+H]⁺.

¹H NMR (DMSO-d₆, 400 MHz) δ 1.14-1.24 (9H), 3.32-3.38 (1H), 4.05-4.21(4H), 4.84-4.26 (1H), 5.14 (1H), 5.68-5.70 (1H), 6.07-6.13 (1H),6.23-6.25 (1H), 7.16-7.24 (5H), 7.34-7.39 (2H), 7.59-7.61 (1H).

Example 3

(2S)-Isopropyl2-(((((2S,4S)-4-(4-amino-2-oxopyrimidin-1(2H)-yl)-1,3-dioxolan-2-yl)methoxy)(phenoxy)phosphoryl)amino)propanoate(3)

Troxacitabine (50 mg, 0.23 mmol) was reacted with the phosphorylatingagent I-38 (0.24 g, 0.58 mmol) according to the procedure described inExample 1 step c, which gave the title compound (40 mg, 35%) as a solid.MS (APCI) 481.0 [M−H]⁻. ¹H NMR (DMSO-d₆, 400 MHz) δ 1.14-1.20 (9H),3.76-3.77 (1H), 4.10-4.18 (2H), 4.22-4.25 (2H), 4.84-4.87 (1H),5.17-5.186 (1H), 5.69-5.70 (1H), 6.03-6.08 (1H), 6.24-6.26 (1H),7.17-7.25 (5H), 7.36-7.40 (2H), 7.62-7.64 (1H).

Example 4

(2S)-Isopropyl2-(((((2S,4S)-4-(4-amino-2-oxopyrimidin-1(2H)-yl)-1,3-dioxolan-2-yl)methoxy)(phenoxy)phosphoryl)amino)propanoate(4)

Troxacitabine (50 mg, 0.23 mmol) was reacted with the phosphorylatingagent I-37 (0.33 g, 0.58 mmol) according to the procedure described inExample 1 step c, which gave the title compound (30 mg, 22%) as a solid.MS (APCI) 599.47 [M+H]⁺.

The compounds listed in TABLE 2 were prepared as pure diastereomersaccording to the procedure described in Example 1 step c using theappropriate intermediate, I-# dia-1 or I-# dia-2.

TABLE 2

diastereomer 1 diastereomer 2 Ex. Interm. R¹⁵ R¹⁶ Ar Yield MS [M + H]⁺Yield MS [M + H]⁺  5 I-40 methyl 2-propyl 1-naphthyl 25% 533.40 33%533.36  6 I-39 methyl cyclohexyl 1-naphthyl 19% 573.35 22% 573.2   7I-41 benzyl cyclohexyl 4-Br-phenyl 18% na 18% na  8 I-6  methyl2-propyl-pentyl phenyl 37% 553.2  35% 553.2   9 I-44 methyl benzyl1-naphthyl 25% 581.2  30% 581.2  10 I-42 methyl 2-propyl2-cyclopropyl-phenyl 34% 523.2  27% 523.2  11 I-43 methyl 2-butyl4-(trimethyl-silyl)-phenyl 37% 569.2  37% 569.2 

Similarly, the compounds listed in TABLE 3 were prepared as purediastereomers according to the procedure described in Example 1 step cusing the appropriate intermediates.

TABLE 3

NMR and MS data were recorded for all exemplified compounds confirmingtheir structures.

Example 35

(2S)-isopropyl2-(((((2S,4S)-4-(2-oxo-4-palmitamidopyrimidin-1(2H)-yl)-1,3-dioxolan-2-yl)methoxy)(phenoxy)phosphoryl)amino)propanoate(35 dial & 35 dia-2)

Compound 2 and 3 were each acylated with palmitic anhydride according tothe method described in WO2008/030373, which gave title compounds.

Example 36

(2S)-methyl2-(((((2S,4S)-4-(2-oxo-4-palmitamidopyrimidin-1(2H)-yl)-1,3-dioxolan-2-yl)methoxy)(phenoxy)phosphoryl)amino)propanoate(36)

Compound 27 dia-2 was acylated with palmitic anhydride according to themethod described in WO2008/030373, which gave title compound.

Comparative Example

Step a)(2S)-2-((bis(4-methoxyphenyl)(phenyl)methyl)amino)-N-(2-oxido-1,3,2-oxathiaphospholan-2-yl)propanamide

To an ice-cold solution of(S)-2-((bis(4-methoxyphenyl)(phenyl)methyl)amino)propanamide (1.40 g,3.58 mmol) and triethylamine (0.60 ml, 4.30 mol) in dichloromethane (8ml) under nitrogen was added dropwise a solution of2-chloro-1,3,2-oxathiaphospholane (0.542 g, 3.80 mmol). The reaction wasallowed to attain room temperature and stirred over the week-end. Thesolution was cooled to 0° C. and a solution of(tert-butylperoxy)trimethylsilane (1.16 g, 7.17 mmol) in heptane wasadded slowly. The reaction mixture was stirred for 90 min, thenconcentrated in vacuum. The residue was suspended in ethyl acetate (10mL), hydrochloride salts were removed by filtration and the solvent wasremoved in vacuum. The residue was dissolved in dry acetonitrile (10 mL)and the resulting solution used in the following step without furtherpurification. Quantitative yield and 80% purity based on ³¹P-NMR wereassumed.

Step b)((2S,4S)-4-(4-amino-2-oxopyrimidin-1(2H)-yl)-1,3-dioxolan-2-yl)methylhydrogen((S)-2-((bis(4-methoxyphenyl)(phenyl)methyl)amino)propanoyl)phosphoramidate

DMAP (229 mg, 1.88 mmol) was added under nitrogen to a solution ofCompound Tr-9 (100 mg, 0.469 mmol) in dry pyridine (5 mL), followed byslow addition of a solution of(2S)-2-((bis(4-methoxyphenyl)(phenyl)methyl)amino)-N-(2-oxo-1,3,2-oxathiaphospholanyl)propanamide (361 mg, 0.563 mmol) in dry acetonitrile (2mL). The resulting solution was stirred at RT under nitrogen for 46 h,then concentrated. The residue was purified by preparative HPLC on aGemini-NX 5 m C18 (100×30 mm) using a gradient from 20% B to 80% B in 17min and a flow of 35 mL/min. Solvent A: 95% water, 5% acetonitrile (10mM in ammonium acetate); Solvent B: 10% water, 90% acetonitrile (10 mMin ammonium acetate). Fractions containing the product were combined andfreeze dried which gave the title compound (80 mg, 26%). MS (ES+) 664.26[M+H]⁺.

Step c)((2S,4S)-4-(4-amino-2-oxopyrimidin-1(2H)-yl)-1,3-dioxolan-2-yl)methylhydrogen ((S)-2-aminopropanoyl)phosphoramidate

Water (50 mL) was added to a solution of the compound from the previousstep (80.5 mg, 0.121 mmol) in dichloromethane followed by addition ofacetic acid (500 mL). The solution was stirred at rt for 12 min, thenTFA (75 mL) was added and the resulting solution was stirred at RT for 5min, diluted with toluene (10 mL), concentrated to dryness and driedunder vacuum. The residue was taken into water containing 10%acetonitrile (10 mL) and washed with tert-butyl methyl ether containing10% hexanes (2×10 mL). The aqueous layer was collected and freeze driedovernight to yield the desired product as the bis-TFA salt (80 mg)having a purity of ˜75% according to LC-MS. The obtained residue wasfurther purified by preparative HPLC on a Hypercarb (21.2×100 mm, I=271nm), using a gradient from 0% to 35% acetonitrile in water. Fractionscontaining the product were combined and freeze dried. MS (ES+) 364.10[M+H]⁺. The structure was confirmed by ¹H and ¹³C NMR.

NMR data for a selection of the exemplified compounds:

Compound 8 dia-1

¹H NMR (DMSO-d₆, 400 MHz) δ 0.81-0.84 (6H), 1.20-1.22 (11H), 1.59 (1H),3.82-3.97 (3H), 4.08-4.16 (2H), 4.22-4.23 (2H), 5.16 (1H), 5.67-5.69(1H), 6.05-6.10 (1H), 6.23-6.24 (1H), 7.16-7.23 (m, 5H), 7.34-7.38 (m,2H), 7.60-7.62 (m, 1H).

Compound 8 dia-2

¹H NMR (DMSO-d₆, 400 MHz) δ 0.81-0.84 (6H), 1.22-1.27 (11H), 1.57 (1H),3.81-3.89 (2H), 3.95-3.98 (1H), 4.05-4.07 (1H), 4.10-4.20 (3H), 5.128(1H), 5.68-5.69 (1H), 6.13-6.14 (1H), 6.22-6.24 (1H), 7.16-7.21 (5H),7.34-7.38 (2H), 7.58-7.60 (1H).

Compound 9 dia-1

³¹P NMR (DMSO-d₆) δ 4.354.

¹H NMR (DMSO-d₆, 400 MHz) δ 1.24-1.26 (3H), 3.98-4.01 (1H), 4.12-4.14(2H), 4.27-4.29 (2H), 5.00-5.08 (2H), 5.16-5.18 (1H), 5.64-5.66 (2H),6.25-6.27 (1H), 6.34 (1H), 7.17-7.22 (2H), 7.31-7.33 (5H), 7.45-7.46(2H), 7.55-7.59 (2H), 7.63-7.64 (1H), 7.74-7.77 (1H), 7.95-7.97 (1H),8.08-8.11 (1H).

Compound 9 dia-2

³¹P NMR (DMSO-d₆) δ 4.159.

¹H NMR (DMSO-d₆, 400 MHz) δ 1.25-1.26 (3H), 3.97-4.01 (1H), 4.08-4.16(2H), 4.23-4.29 (2H), 5.04-5.16 (3H), 5.65-5.66 (1H), 6.26 (1H),6.36-6.42 (1H), 7.17-7.24 (2H), 7.326 (5H), 7.41-7.49 (2H), 7.57-7.64(3H), 7.74-7.76 (1H), 7.95-7.97 (1H), 8.10-8.12 (1H).

Compound 11-dia-1

¹H NMR (DMSO-d₆, 400 MHz) δ 0.23 (9H), 0.78-0.82 (3H), 1.08-1.12 (3H),1.20-1.22 (3H), 1.44-1.49 (2H), 3.77-3.79 (1H), 4.09-4.23 (4H),4.67-4.72 (1H), 5.16-5.16 (1H), 5.69-5.70 (1H), 6.04-6.10 (1H),6.23-6.25 (1H), 7.15-7.24 (4H), 7.48-7.50 (2H), 7.61-7.63 (1H).

Compound 11 dia-2

¹H NMR (DMSO-d₆, 400 MHz) δ 0.22-0.24 (9H), 0.78-0.82 (3H), 1.10-1.11(3H), 1.22-1.24 (3H), 1.46-1.50 (2H), 4.05-4.07 (1H), 4.11-4.22 (4H),4.70-4.71 (1H), 5.14 (1H), 5.69-5.71 (1H), 6.07-6.11 (1H), 6.23-6.25(1H), 7.16-7.24 (4H), 7.49-7.51 (2H), 7.60-7.62 (1H).

For a prodrug to be liver targeted, a correct processing of the prodrugis crucial. The prodrug should be stable in intestinal fluid, andprocessed in the liver by liver enzymes in a first pass metabolism toform the monophosphate. The formed monophosphate is then to beanabolized by cellular kinases in the hepatocytes to the activetriphosphate species. Additionally, the anti-cancer drug should be toxicto proliferating cells. Suitable methods to evaluate compounds for theseproperties are, for example, as set out below.

Stability in human intestinal S9 fraction (HIS9) and in human liver S9fraction (HLS9),

Stock solutions of each test compound (10 mM) were prepared in DMSO andstored at −20° C. Prior to the start of the experiment, the testcompounds were diluted to 500 μM in 50% acetonitrile in water. Thereaction mixture was prepared in a total volume of 250 μL containing 5mM MgCl₂, 1 mM NADPH and 5 μM test compound in 50 mM potassium phosphatebuffer (pH 7.4). The reaction was initiated by addition of human liveror intestinal S9 fraction with a final concentration of 0.4 mgprotein/mL (Xeno Tech). The reaction mixture was incubated on an orbitalshaker at 37° C. At the desired time points (0, 10, 30 and 60 minutes)aliquots of 50 μL were taken and the reaction was stopped by mixing with150 μL acetonitrile containing internal standard. Standard solutions ofeach test compound were prepared from the 500 μM solution by dilutingthe solution to a final concentration of 5 μM in boiled human S9 (0.4 mgprotein/mL), 5 mM MgCl₂ and 50 mM potassium phosphate buffer (pH 7.4).The standards and samples were kept on ice for 30 min then centrifugedat 3 000 g for 20 minutes at 10° C., there after 10 μL of supernatantwas mixed with 200 μL 50% acetonitrile in water. 0.5 μM of each testcompound in 50% acetonitrile in water was injected into the LC/MS-MS todetermine the daughter ion, declustring potential (DP), collision energy(CE) and collision cell exit potential (CXP) in order to develop aLC/MS-MS method. The compounds were separated using a C18 column with aQTRAP5500 system. The mobile phase consisted of solvent A (98% water, 2%acetonitrile, 0.1% acetic acid or 10 mM ammonium acetate) and solvent B(80% acetonitrile, 20% water, 0.1% acetic acid or 10 mM ammoniumacetate). Elution of the compounds was performed by using a gradient ofsolvent B from 0% to 100%. 5 μL of standard points and samples wereinjected for analysis with QTRAP5500.

The amount of parent compound was determined on the basis of the peakarea for each time point compared to standard which was set to 5 μM.Intrinsic clearance (CL_(int)) and half-life (t_(1/2)) were determinedfrom the disappearance curves of the test compound using Excel software.

Cell Cytotoxicity Assays

Cells were seeded 24 hours prior to compound addition. Each testcompound (serially diluted from 100 μM) was added to Huh7 (1.5×10⁴cells/well) or HepG2 (1.5×10⁴ cells/well), and allowed to incubate for 5days at 37° C. A medium only control was used to determine the minimumabsorbance value and an untreated cell value. At the end of the growthperiod, XTT dye from Polysciences Europe GmbH was added to each well.The absorbance at 450 nm with a reference wavelength of 600 nM was readwith a Sunrise (Tecan) using the medium only control wells as blanks.The 50% inhibition value (CC₅₀) was determined by comparing the degreeof inhibition (compared to cell control) plotted against compoundconcentration. Results from the dilution series were fitted to asigmoidal dose-response curve.

Compounds of the invention were evaluated in these assays to assess thestability in human intestinal S9 fraction (HIS9) and human liver S9fraction (HLS9), and for Cell Cytotoxicity in HUH7, HEP3B and HEPG2cells. The results are summarised in TABLE B1.

TABLE B1 HUH7 HEP3B HEPG2 CL_(int) CL_(int) CC₅₀ CC₅₀ CC₅₀ Liver S9Intestinal S9 Example (μM) (μM) (μM) (μL/min/mg) (μL/min/mg)  1 >100na >100 12 6  2 1.75 na 0.248 13 6  3 3.28 na 0.371 8 6  4 12.0 na 0.93684 123  4 dia-2 1.55 na 0.093 38 18  5 dia-1 0.465 na 0.107 32 21  5dia-2 0.602 na 0.114 31 13  6 dia-1 0.258 na 0.092 91 36  6 dia-2 0.316na 0.048 61 25  7 dia-1 1.02 na 0.24 148 147  7 dia-2 0.134 na 0.058 6027  8 dia-1 0.123 na 0.007 130 86  8 dia-2 0.074 0.035 0.017 143 25  9dia-1 0.164 na 0.023 133 171  9 dia-2 0.158 na 0.016 94 127 10 dia-10.392 na 0.062 26 12 10 dia-2 0.556 na 0.051 22 14 11 dia-1 0.026 0.0180.054 51 6 11 dia-2 na 0.031 0.054 81 28 12 dia-1 4.33 na 0.481 182 30012 dia-2 5.02 na 1.09 97 300 13 diamix 0.663 na 0.163 85 27 4:1 14 dia-10.216 na 0.016 88 30 14 dia-2 0.200 na 0.012 159 59 15 dia-1 0.025 na0.037 167 87 15 dia-2 0.026 na 0.019 95 36 16 dia-1 1.20 0.106 0.151 508 16 dia-2 0.152 0.053 0.130 59 8 17 dia-1 50.0 na 50.0 6 6 17 dia-250.0 na 50.0 6 6 18 dia-1 0.461 0.228 0.248 21 6 18 dia-2 0.076 0.1130.065 30 7 19 dia-1 0.091 na 0.018 19 26 19 dia-2 0.071 0.058 0.014 2417 20 dia-1 0.216 na 0.074 45 21 20 dia-2 0.073 0.078 0.060 25 6 21dia-1 0.574 na 0.163 61 29 21 dia-2 0.070 na 0.048 22 10 22 dia-1 0.033na 0.012 49 52 22 dia-2 0.040 na 0.011 43 34 23 dia-1 na 0.01  0.0086186 32 23 dia-2 na na na 300 20 24 dia-1 na na na na na 24 dia-2 na nana na na 25 dia-1 na na na na na 25 dia-2 na na na na na 26 dia-1 na4.34  1.21 7 6 26 dia-2 4.73 4.02  1.06 10 6 Troxa- 0.646 0.279 0.218 nana citabine 27 dia-1 1.44 na 0.151 38 11 27 dia-2 1.02 0.348 0.223 57 628 dia-2 15.6 na 2.72 20 40 29 dia-2 0.495 0.075 na 36 18 30 dia-2 na nana 120 11 31 dia-2 na na na 8 6 32 dia-2 na na na 27 8 33 dia-1 na na na180 27 33 dia-2 na na na 230 75 34 dia-2 0.524 0.210 0.236 64 6 35 dia-20.011 na 0.007 34 51 36 0.009 0.019 na na na = not availablenaTriphosphate Formation Assay

Each compound was tested in triplicates in the assay.

Fresh human plated hepatocytes (Biopredic, France) in 12-well plateswere used. Each well was plated with 0.76×10⁶ cells and incubated with a10 μM DMSO solution of compound (0.1% DMSO) in 1 mL incubation medium ina CO₂ incubator at 37° C. for 8 hours. Huh7 cells grown in DMEM withantibiotics and 10% fetal calf serum were seeded in 12 well plates,2×10⁶ cells/well. After 24 hrs 1 mL of 10 μM compound in medium wasadded and the cells were incubated another 6-8 hrs.

The incubation was stopped by washing each well with 1 mL ice coldHank's balanced solution, pH 7.2 twice, followed by addition of 0.5 mLice cold 70% methanol. Immediately after the addition of methanol, thecell-layer was detached from the bottom of the well by a cell scraperand sucked up and down 5-6 times with an automatic pipet. The cellsuspension was transferred to a glass vial and stored over night at −20°C.

The samples, each consisting of various levels of prodrug, freenucleoside, and mono-, di- and triphosphate were then vortexed andcentrifuged at 10° C. for 10 minutes, at 14000 rpm in an Eppendorfcentrifuge 5417R. The supernatants were transferred to 2 mL glass vialswith insert and subjected to bioanalysis as follows:

An internal standard (Indinavir) was added to each sample and thesamples (10 μL injection volume) were analysed on a two column systemcoupled to a QTRAP 5000 mass spectrometer. The two column systemconsisted of two binary pumps, X and Y, two switching valves and anautosampler. The two HPLC columns used were a Synergy POLAR-RP 50*4.6mm, 4 μm particles and a BioBasic AX 50*2.1 mm 5 μm particles. The LCflow rates were 0.4-0.6 mL/min mL/min (the higher flow rate were used inthe recondition step).

The HPLC mobile phases for the POLAR-RP column consisted of 10 mmol/Lammonium acetate in 2% acetonitrile (mobile phase A) and 10 mmol/Lammonium acetate in 90% acetonitrile (mobile phase B) and for theBioBasic AX column 10 mmol/L ammonium acetate in 2% acetonitrile (mobilephase C) and 1% ammonium hydroxide in 2% acetonitrile (mobile phase D).The HPLC gradient for pump Y started at 0% mobile phase B and was heldfor 2 min. During loading phase, the mobile phase went through thePOLAR-RP and BioBasic AX column, and prodrug, nucleoside and internalstandard were trapped on the POLAR-RP column; whereas the nucleotides(mono-, di- and triphosphates) eluted on to the BioBasic AX column andwere trapped there.

In the next step, the flow was switched from the POLAR-RP column to theMS and the mobile phase C switched from pump X to the BioBasic AXcolumn. The compounds on the POLAR-RP column were eluted with a gradientfrom 0% B up to 100% B in about two minutes and analyzed in positive ornegative mode using the multiple reaction monitoring mode (MRM). In thelast step the flow from the BioBasic AX column was switched to the MSand the phosphates were eluted with a of about 7 minutes gradient up 50%D, and analyzed in positive or negative mode using MRM. During the laststep both columns are reconditioned. Triphosphate concentration for eachcompound was then determined by comparison with standard curves whichwere made by analysis of standard samples with known concentrations oftriphosphate. The standards were run in the same matrices as the testsamples. Due to variations in phosphorylation levels between hepatocytedonors, an internal reference compound is required in each run of theassay in order to enable ranking the results from different runs to eachother.

Throughout the specification and the claims which follow, unless thecontext requires otherwise, the word ‘comprise’, and variations such as‘comprises’ and ‘comprising’, will be understood to imply the inclusionof a stated integer, step, group of integers or group of steps but notto the exclusion of any other integer, step, group of integers or groupof steps.

All documents referred to herein, including patents and patentapplications, are incorporated by reference in their entirety.

The invention claimed is:
 1. A compound represented by formula Ia:

wherein: R¹ is NH₂; R² is H; R¹³ is phenyl, optionally substituted with1, 2 or 3 R²²; R¹⁵ is methyl; R¹⁶ is 2-pentyl; each R²² is independentlyselected from halo, C₁-C₆alkyl, C₂-C₆alkenyl, C₁-C₆haloalkyl,C₁-C₆alkoxy, C₁-C₆haloalkoxy, phenyl, hydroxyC₁-C₆alkyl,C₃-C₆cycloalkyl, C₁-C₆alkylcarbonyl, C₃-C₆cycloalkylcarbonyl,carboxyC₁-C₆alkyl, hydroxy, amino CN, and NO₂, or any two R²² groupsattached to adjacent ring carbon atoms can combine to form—O—(CR²³R^(23′))₁₋₆-O—; R²³ and R^(23′) are independently H orC₁-C₃alkyl; or a pharmaceutically acceptable salt and/or solvatethereof.
 2. A compound according to claim 1 wherein R¹³ is substitutedwith one or two R²².
 3. A compound according to claim 1, wherein R¹³ isunsubstituted phenyl.
 4. A pharmaceutical composition comprising atherapeutically effective amount of a compound according to claim 1 inassociation with a pharmaceutically acceptable adjuvant, diluent orcarrier.
 5. A pharmaceutical combination comprising a therapeuticallyeffective amount of compound according to claim 1, further comprisingone or more additional therapeutic agent(s) selected from the groupconsisting of chemotherapeutical agent, multi-drug resistance reversingagent and biological response modifier.
 6. The pharmaceuticalcombination according to claim 5, wherein the further therapeutic agentis a chemotherapeutical agent.
 7. The compound of claim 1, that is


8. The compound of claim 1, that is


9. The compound of claim 1 that is